Proteins with enhanced functionality and methods of making novel proteins using circular permutation

ABSTRACT

The present disclosure is relates to novel proteins and peptides having novel and/or enhanced functions and/or behaviors with respect to a native protein or peptide, and methods of making the novel proteins and peptides using techniques of circular permutation and protein engineering.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of and is a Continuation-in-Partapplication of International Application No. PCT/US2006/004675, entitled“Novel Proteins with Enhanced Functionality and Methods of Making NovelProteins Using Circular Permutation” filed on Feb. 10, 2006, whichclaims priority to copending U.S. provisional patent application Ser.No. 60/651,850, entitled “Lipase Variants from Candida Antarctica” filedon Feb. 10, 2005; Ser. No. 60/696,325, entitled “Lipase Variants fromCandida Antarctica” filed on Jul. 1, 2005; Ser. No. 60/714,462, entitled“Circularly Permuted Proteins and Methods of Using Circular Permutationto Improve Protein Design and Activity” filed on Sep. 6, 2005; and Ser.No. 60/726,009, entitled “Circularly Permuted Proteins and Methods ofUsing Circular Permutation to Improve Protein Design and Activity” filedon Oct. 12, 2005, each of which are entirely incorporated herein byreference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under grant numberCHE-0404677 awarded by the National Science Foundation. The governmenthas certain rights in the invention.

FIELD OF THE DISCLOSURE

The present disclosure is generally related to novel proteins andpeptides having novel and/or enhanced functions and/or behaviors andmethods of making the novel proteins and peptides.

BACKGROUND

Lipases play an important role in asymmetric biocatalysis. Their broadsubstrate specificity, generally high regio- and enantio-selectivity, aswell as their ability to function in aqueous and organic reaction mediummake them versatile tools for the kinetic resolution, derivatization,chiral synthesis, and polymerization of esters. Lipases can catalyze theformation, hydrolysis, and substitution (transesterification) of esterbonds, amid bonds, and the like. They are important biocatalysts inproduction of chiral building blocks for fine chemicals andpharmaceuticals, as well as in bulk products such as laundry detergent.

Suitable enzymes for a particular substrate can be identified byscreening natural lipases or can be tailored by protein engineering. Inthe latter case, rational protein design, random mutagenesis, and DNAshuffling have generated laboratory catalysts with altered specificity,selectivity, and stability. However, very few natural and lab-madelipases show activity and enantioselectivity for bulky substrates suchas esters of large secondary and tertiary alcohols. It has beenhypothesized that the cause for the poor turnover of these substratesarises from steric constraints in the lipase active site, yet proteinengineers have so far failed to generate improved biocatalysts.Tailoring these enzymes to novel, unnatural substrates is one of theprimary challenges of protein engineering. Circular permutation mayprovide the ability to meet such challenges.

Circular permutation is a technique where the normal termini of apolypeptide are linked and new termini are created by breaking thebackbone elsewhere. In many polypeptides, the normal termini are inclose proximity and can be joined by a short amino acid sequence. Thebreak in the polypeptide backbone can be at any point, preferably at apoint where the function and folding of the polypeptide are notdestroyed. Circular permutation creates new C- and N-termini, so thetechnique is often used in the creation of fusion proteins where thefused peptide or protein is attached at a different place on the hostprotein. For example, if the natural termini are at the interior of thebase protein, it may be disruptive to attach a peptide or protein at thenatural termini. By changing the attachment location to a place near theexterior of the host protein, stability of the host protein may bemaintained.

Circular permutation provides an experimental way to investigate thebiophysical consequences of backbone rearrangement or removal on ligandbinding in ways not available using traditional deletion mutants.Circularly permuted proteins have been used previously to investigatethe protein folding problem (Yang Y, et al. (1993) Proc Natl Acad SciUS. 90:11980-1984; Graf R, et al. (1996) Proc Natl Acad Sci USA93:11591-11596). Both naturally occurring and synthetic circularlypermuted proteins have been identified (Heinemann U, et al. (1995) ProgBiophys Molec Biol 64:122-143; Lindqvist Y, et al. (1997) Curr OpinionStruc Biol 7:422-427; Goldenberg D P, et al. (1983) J Mol Biol164:407-413; Luger K, et al. (1989) Science 243-206-209). U.S. Pat. No.5,635,599 to Pastan et al. discloses fusion proteins created fromcircularly permuted interleukin 4 (IL4).

As mentioned above, circular permutants generally are created bydisrupting the polypeptide chain at a selected point to create newtermini and bridging the two natural termini either directly or througha linker such as an amino acid linker. Circular permutation thus has theeffect of essentially preserving the sequence and identity of the aminoacids of a protein, while generating new termini at different locations.Moreover, the tertiary structure of the protein is generally conserved.Circularly permuted proteins can be made chemically or created byrecombinant techniques.

SUMMARY

Briefly described, embodiments of the present disclosure include novelproteins having novel or improved/enhanced functions or behavior. Inembodiments of the present disclosure, the novel proteins are circularlypermuted proteins having native amino-terminal and carboxy-terminal endsthat have been linked, optionally with a linker sequence, and newamino-terminal and carboxy-terminal ends that are different from thenative amino-terminal and carboxy-terminal ends of a correspondingnative protein. In some preferred embodiments, the circularly permutedproteins include at least one improvement over the corresponding nativeprotein. The improvement can include, but is not limited to, increasedactivity, increased accessibility to the active site, increasedflexibility of the active site, increased the enantioselectivity, andbroader and/or changed substrate specificity.

Embodiments of the circularly permuted proteins of the presentdisclosure also include circularly permuted proteins of theα/β-hydrolase fold family. The circularly permuted proteins of theα/β-hydrolase fold family include original amino-terminal andcarboxy-terminal ends that have been linked, optionally with a linkersequence, and new amino-terminal and carboxy-terminal ends that aredifferent from the original amino-terminal and carboxy-terminal ends ofa corresponding native protein of the α/β-hydrolase fold family. Inpreferred embodiments, the circularly permuted protein of theα/β-hydrolase fold family include at least one improvement over thecorresponding native protein, including but not limited to, increasedactivity, increased accessibility to the active site, increasedflexibility of the active site, increased the enantioselectivity, andbroader or changed substrate specificity.

Some embodiments of the circularly permuted proteins of the presentdisclosure and circularly permuted proteins of the α/β-hydrolase foldfamily also include at least one secondary mutation. In embodiments ofthe disclosure, the secondary mutation is selected from a deletion,insertion, or a substitution of one or more amino acids with differentamino acids, or a combination thereof. The secondary mutation(s) resultin a second circularly permuted protein. In preferred embodiments, thesecond circularly permuted protein has at least one improvement over thecorresponding native protein and the corresponding circularly permutedprotein. The improvements include, but are not limited to, increasedactivity, increased stability, broader or changed substrate specificity,increased active site flexibility, increased enantioselectivity, andcombinations thereof.

The present disclosure also includes methods of making a novel proteinof the α/β-hydrolase fold family. The methods include, but are notlimited to, the following steps: selecting a native protein of theα/β-hydrolase fold family having an active site, an amino-terminal end,and a carboxy-terminal end; linking the amino-terminal andcarboxy-terminal ends of the native protein to form a circular proteinmolecule; creating a library of circularly permuted proteins of theα/β-hydrolase fold family, where at least one circularly permutedprotein in the library is a variant of the native protein having newamino-terminal and carboxy-terminal ends that are different from theamino-terminal and carboxy terminal ends of the native protein;selecting functional variants from the library; and testing selectedfunctional variants for improvements with respect to the native protein.Such improvements include, but are not limited to, increased activity,increased accessibility, increased enantioselectivity, increasedflexibility of the active site, increased stability, broader and/orchanged substrate specificity, and combinations thereof.

Methods of the present disclosure also include methods of making a novelprotein including the following steps: selecting a native protein havingan active site, an amino-terminal end, and a carboxy-terminal end;linking the amino-terminal and carboxy-terminal ends of the nativeprotein to form a circular protein molecule; creating a library ofcircularly permuted proteins, where at least one circularly permutedprotein in the library is a variant of the native protein having newamino-terminal and carboxy-terminal ends that are different from theamino-terminal and carboxy terminal ends of the native protein;selecting functional variants from the library; mapping the location ofthe new amino-terminal and carboxy-terminal ends in the functionalvariants to determine locations of permissible permutations; selectingfunctional variants having new amino-terminal and carboxy-terminal endslocated near a binding site of the protein; and testing selectedfunctional variants for improvements with respect to the native protein,wherein the improvement is selected from: increased activity, increasedaccessibility, increased enantioselectivity, increased stability, andbroader or changed substrate specificity.

The methods of making novel circularly permuted proteins of the presentdisclosure described above also include performing secondary engineeringon one or more selected functional variants to produce at least onesecondary circular permuted protein. In some embodiments, the secondaryengineering include introducing at least one secondary mutation into thecircularly permuted protein, where the secondary mutation includes, butis not limited to, deletion, insertion, and/or substitution of one ormore amino acids of the circularly permuted protein, or a combinationthereof. The secondary mutation(s) result in a second circularlypermuted protein. In preferred embodiments, the second circularlypermuted protein has at least one improvement over the correspondingnative protein and the corresponding circularly permuted protein. Theimprovements include, but are not limited to, increased activity,increased stability, broader or changed substrate specificity, increasedactive site flexibility, increased enantioselectivity, and combinationsthereof.

Other aspects, compositions, methods, features, and advantages of thepresent disclosure will be or become apparent to one with skill in theart upon examination of the following drawings and detailed description.It is intended that all such additional compositions, methods, features,and advantages be included within this description, be within the scopeof the present disclosure, and be protected by the accompanying claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The disclosure can be better understood with reference to the followingdrawings. The components in the drawings are not necessarily to scale,emphasis instead being placed upon clearly illustrating the principlesof the present disclosure.

FIG. 1A illustrates the concept of circular permutation, showing anative protein structure on the left, and three circular permutants ofthe native protein on the right. FIG. 1B illustrates the process ofcircular permutation using recombinant DNA.

FIG. 2A illustrates a schematic of the secondary structural elements ofproteins of the α/β-hydrolase fold family. FIGS. 2B and 2C illustratethe secondary and tertiary structure of two members of α/β-hydrolasefold family, lipase B from Candida antarctica (CALB) and the epoxidehydrolase from Agrobacterium radiobacter, respectively.

FIG. 3 is a circular permutation diagram of CALB illustrating thedistribution of the termini location of 89 randomly chosen librarymembers (outer circle) (library size ˜0.5×10⁶).

FIG. 4 shows the screening technique used to identify functionalvariants of the CALB library. Screening was performed on tributyrinplates to assess for hydrolase activity. Both a primary and secondaryscreening were performed, as illustrated.

FIG. 5 is a circular permutation diagram of CALB illustrating thedistribution of the termini location of 63 functional library memberswith unique sequences (outer circle).

FIG. 6 illustrates the structure of CALB and identifies the locations ofpermissible permutation sites (indicated by hatched areas) and thevariants selected for further characterization (indicated by amino acidlocation of the new amino terminus).

FIG. 7 is a far-UV circular dichroism spectra for CALB variants with newtermini in helix 7/9. The insert is a graph of thermostability data forthe same variants.

FIG. 8 is a far-UV circular dichroism spectra for CALB variants with newtermini in helix 16/17. The insert is a graph of thermostability datafor the same variants FIGS. 9A-B are schematic diagrams of the regionrepresenting the amino an carboxy-termini in native CALB and thelocation of the external loop in variant cp283.

FIG. 9A shows the wild type CALB termini. FIG. 9B illustratesincremental truncation of the C-terminal tail in wild type CALB. FIG. 9Cdepicts the external loop in cp283.

FIG. 9D illustrates incremental deletions/truncations of the externalloop structure in cp283. On the right-hand side of FIG. 9, severalpartial sequences corresponding to either wild type CALB (or C-terminaltruncations thereof) or cp283 (or loop truncations thereof) areillustrated.

FIG. 10 is a graph illustrating gel filtration analysis of cpCALBvariants.

FIG. 11 is a graph illustrating an analysis of the secondary structureof the cpCALB variants.

FIG. 12 illustrates Table 2.

FIG. 13 illustrates Table 3.

DETAILED DESCRIPTION

Embodiments of the present disclosure will employ, unless otherwiseindicated, conventional techniques of synthetic organic chemistry,biochemistry, molecular biology, and the like, which are within theskill of one in the art. Such techniques are explained fully in theliterature.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how toperform the methods and use the compositions disclosed and claimedherein. Efforts have been made to ensure accuracy with respect tonumbers (e.g., amounts, temperature, etc.) but some errors anddeviations should be accounted for. Unless indicated otherwise, partsare parts by weight, temperature is in ° C., and pressure is at or nearatmospheric. Standard temperature and pressure are defined as 20° C. and1 atmosphere.

Before the embodiments of the present disclosure are described indetail, it is to be understood that unless otherwise indicated thepresent disclosure is not limited to particular materials, reagents,reaction materials, manufacturing processes, or the like, as such mayvary. It is also to be understood that the terminology used herein isfor purposes of describing particular embodiments only, and is notintended to be limiting. It is also possible in the present disclosurethat steps may be executed in different sequence where this is logicallypossible.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an” and “the” include plural referentsunless the context clearly dictates otherwise. Thus, for example,reference to “a support” includes a plurality of supports. In thisspecification and in the claims that follow, reference will be made to anumber of terms that shall be defined to have the following meanings,unless a contrary intention is apparent.

Definitions:

“Circular permutation,” as used herein, refers to the process of takinga straight-chain molecule, fusing the ends (directly or through alinker) to form a circular molecule, and then cutting the circularmolecule at a different location to form a new straight chain moleculewith different termini. Circular permutation also includes any processthat results in a circularly permutated protein, as defined herein.Circular permutation thus preserves the sequence and identity of theamino acids of a protein, while generating new termini at differentlocations.

The term “circularly permuted,” “circularly permuted protein,” andvariations thereof as used herein refers to DNA, RNA and protein,essentially any linear molecule, in which the termini have been joinedtogether, either directly or through a linker, to produce a circularmolecule, and then the circular molecule is opened at another locationto produce a new linear molecule with termini different from the terminiin the original native/molecule. Circular permutations include thosemolecules whose structure is equivalent to a molecule that has beencircularized and then opened. Thus, a circularly permuted molecule maybe synthesized de novo as a linear molecule and never go through acircularization and opening step. The particular circular permutation ofa protein or peptide, or a polynucleotide encoding such protein orpeptide, is designated by the prefix “cp” (for “circular permutation”)followed by the residue number of the amino acid where the N-terminusnow resides in the circularly permuted polypeptide. Thus, thedesignation cp44 designates a circularly permuted protein in which thenew N-terminus (e.g., in the position following the new opening site orwhere a peptide bond has been eliminated) is at amino acid 44 of theunpermuted or wild type protein.

The terms “unpermuted,” “native,” “wild type”, or “unmodified”polypeptide, protein or enzyme, are used herein to provide a referencepoint for the polypeptide, protein, or enzyme prior to its rearrangementinto a circularly permuted molecule, as described above. Typically, theunmodified, native, or wild type polypeptide, protein, or enzyme has anamino acid sequence that correspond substantially to the amino acidsequence of the polypeptide, protein, or enzyme as it generally occursnaturally or in vivo.

The term “linker” or “linker sequence,” as used herein, refers to amolecule that is used to join the amino and carboxyl termini of aprotein or its corresponding nucleic acid sequence (e.g. the RNA or DNAmolecule encoding the protein). The linker is capable of formingcovalent bonds to both the amino and carboxyl terminus. Suitable linkersare well known to those of skill in the art and include, but are notlimited to, straight or branched-chain carbon linkers, heterocycliccarbon linkers, or peptide linkers. The linkers may be joined to thecarboxyl and amino terminal amino acids through their side groups (e.g.,through a disulfide linkage to cysteine). However, in a preferredembodiment, the linkers will be joined to the alpha carbon amino andcarboxyl groups of the terminal amino acids. Another method for linkingthe wild type termini of a protein is the direct connection between thenative amino and carboxylate moieties. The term “linker” may also referto the nucleic acid sequence corresponding to the linking peptidesequence. In some embodiments, the circularly permuted protein isproduced by linking the ends of the corresponding DNA or RNA sequence,forming various permutants by cutting the circularized nucleic acidsequence, and subsequently translating the nucleic acid sequences toform the circularly permuted protein(s).

The term “residue” as used herein refers to an amino acid that isincorporated into a peptide. The amino acid may be a naturally occurringamino acid and, unless otherwise limited, may encompass known analogs ofnatural amino acids that can function in a similar manner as naturallyoccurring amino acids.

The term “opening site,” as used herein when referring to circularpermutation, refers to the position at which a peptide bond would beeliminated to form new amino and carboxyl termini. The opening site isdesignated by the positions of the pair of amino acids, located betweenthe amino and carboxyl termini of the unpermuted (native) protein, thatbecome the new amino and carboxyl termini of the circularly permutedprotein.

As used herein, “polynucleotides” include single or multiple strandedconfigurations, where one or more of the strands may or may not becompletely aligned with another. The terms “polynucleotide” and“oligonucleotide” shall be generic to polydeoxynucleotides (containing2-deoxy-D-ribose), to polyribonucleotides (containing D-ribose), to anyother type of polynucleotide which is an N-glycoside of a purine orpyrimidine base, and to other polymers in which the conventionalbackbone has been replaced with a non-naturally occurring or syntheticbackbone or in which one or more of the conventional bases has beenreplaced with a non-naturally occurring or synthetic base. An“oligonucleotide” generally refers to a nucleotide multimer of about 2to 100 nucleotides in length, while a “polynucleotide” includes anucleotide multimer having any number of nucleotides greater than 1,although they are often used interchangeably.

A “nucleotide” refers to a sub-unit of a nucleic acid (whether DNA orRNA or analogue thereof) which includes a phosphate group, a sugar groupand a nitrogen containing base, as well as analogs of such sub-units.

A “nucleoside” references a nucleic acid subunit including a sugar groupand a nitrogen containing base. It should be noted that the term“nucleotide” is primarily used herein to describe embodiments of thedisclosure, but that one skilled in the art would understand that theterm “nucleoside” and “nucleotide” are interchangeable in mostinstances. One skilled in the art would have the understanding thatadditional modification to the nucleoside may be appropriate, and oneskilled in the art has such knowledge.

A “nucleoside moiety” refers to a molecule having a sugar group and anitrogen containing base (as in a nucleoside) as a portion of a largermolecule, such as in a polynucleotide, oligonucleotide, or nucleosidephosphoramidite.

A “nucleotide monomer” refers to a molecule which is not incorporated ina larger oligo- or poly-nucleotide chain and which corresponds to asingle nucleotide sub-unit; nucleotide monomers may also have activatingor protecting groups, if such groups are necessary for the intended useof the nucleotide monomer.

It will be appreciated that, as used herein, the terms “nucleoside” and“nucleotide” will include those moieties which contain not only thenaturally occurring purine and pyrimidine bases, e.g., adenine (A),thymine (T), cytosine (C), guanine (G), or uracil (U), but also modifiedpurine and pyrimidine bases and other heterocyclic bases which have beenmodified (these moieties are sometimes referred to herein, collectively,as “purine and pyrimidine bases and analogs thereof”). Suchmodifications include, e.g., diaminopurine and its derivatives, inosineand its derivatives, alkylated purines or pyrimidines, acylated purinesor pyrimidines, thiolated purines or pyrimidines, and the like, or theaddition of a protecting group such as acetyl, difluoroacetyl,trifluoroacetyl, isobutyryl, benzoyl, 9-fluorenylmethoxycarbonyl,phenoxyacetyl, dimethylformamidine, N,N-diphenyl carbamate, or the like.The purine or pyrimidine base may also be an analog of the foregoing;suitable analogs will be known to those skilled in the art and aredescribed in the pertinent texts and literature. Common analogs include,but are not limited to, 1-methyladenine, 2-methyladenine,N6-methyladenine, N6-isopentyladenine, 2-methylthio-N6-isopentyladenine,N,N-dimethyladenine, 8-bromoadenine, 2-thiocytosine, 3-methylcytosine,5-methylcytosine, 5-ethylcytosine, 4-acetylcytosine, 1-methylguanine,2-methylguanine, 7-methylguanine, 2,2-dimethylguanine, 8-bromoguanine,8-chloroguanine, 8-aminoguanine, 8-methylguanine, 8-thioguanine,5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,5-ethyluracil, 5-propyluracil, 5-methoxyuracil, 5-hydroxymethyluracil,5-(carboxyhydroxymethyl)uracil, 5-(methylaminomethyl)uracil,5-(carboxymethylaminomethyl)-uracil, 2-thiouracil,5-methyl-2-thiouracil, 5-(2-bromovinyl)uracil, uracil-5-oxyacetic acid,uracil-5-oxyacetic acid methyl ester, pseudouracil,1-methylpseudouracil, queosine, inosine, 1-methylinosine, hypoxanthine,xanthine, 2-aminopurine, 6-hydroxyaminopurine, 6-thiopurine, and2,6-diaminopurine.

An “internucleotide bond” refers to a chemical linkage between twonucleoside moieties, such as a phosphodiester linkage in nucleic acidsfound in nature, or such as linkages well known from the art ofsynthesis of nucleic acids and nucleic acid analogues. Aninternucleotide bond may include a phospho or phosphite group, and mayinclude linkages where one or more oxygen atoms of the phospho orphosphite group are either modified with a substituent or replaced withanother atom, e.g., a sulfur atom, or the nitrogen atom of a mono- ordi-alkyl amino group.

The term “polypeptides” and “protein” include proteins and fragmentsthereof. Polypeptides are disclosed herein as amino acid residuesequences. Those sequences are written left to right in the directionfrom the amino to the carboxy terminus. In accordance with standardnomenclature, amino acid residue sequences are denominated by either athree letter or a single letter code as indicated as follows: Alanine(Ala, A), Arginine (Arg, R), Asparagine (Asn, N), Aspartic Acid (Asp,D), Cysteine (Cys, C), Glutamine (Gln, Q), Glutamic Acid (Glu, E),Glycine (Gly, G), Histidine (His, H), Isoleucine (Ile, I), Leucine (Leu,L), Lysine (Lys, K), Methionine (Met, M), Phenylalanine (Phe, F),Proline (Pro, P), Serine (Ser, S), Threonine (Thr, T), Tryptophan (Trp,W), Tyrosine (Tyr, Y), and Valine (Val, V).

“Variant” refers to a polypeptide that differs from a referencepolypeptide, but retains essential properties. A typical variant of apolypeptide differs in amino acid sequence from another, referencepolypeptide. Generally, differences are limited so that the sequences ofthe reference polypeptide and the variant are closely similar overalland, in many regions, identical. A variant and reference polypeptide maydiffer in amino acid sequence by one or more modifications (e.g.,substitutions, additions, and/or deletions). A substituted or insertedamino acid residue may or may not be one encoded by the genetic code. Avariant of a polypeptide may be naturally occurring such as an allelicvariant, or it may be a variant that is not known to occur naturally. Inaddition, the term “variant” as used herein includes circularpermutations of proteins and peptides.

Modifications and changes can be made in the structure of thepolypeptides of in disclosure and still obtain a molecule having similarcharacteristics as the polypeptide (e.g., a conservative amino acidsubstitution). For example, certain amino acids can be substituted forother amino acids in a sequence without appreciable loss of activity.Because it is the interactive capacity and nature of a polypeptide thatdefines that polypeptide's biological functional activity, certain aminoacid sequence substitutions can be made in a polypeptide sequence andnevertheless obtain a polypeptide with like properties.

In making such changes, the hydropathic index of amino acids can beconsidered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a polypeptide is generallyunderstood in the art. It is known that certain amino acids can besubstituted for other amino acids having a similar hydropathic index orscore and still result in a polypeptide with similar biologicalactivity. Each amino acid has been assigned a hydropathic index on thebasis of its hydrophobicity and charge characteristics. Those indicesare: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine(+2.8); cysteine/cysteine (+2.5); methionine (+1.9); alanine (+1.8);glycine (−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9);tyrosine (−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5);glutamine (−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9);and arginine (−4.5).

It is believed that the relative hydropathic character of the amino aciddetermines the secondary structure of the resultant polypeptide, whichin turn defines the interaction of the polypeptide with other molecules,such as enzymes, substrates, receptors, antibodies, antigens, and thelike. It is known in the art that an amino acid can be substituted byanother amino acid having a similar hydropathic index and still obtain afunctionally equivalent polypeptide. In such changes, the substitutionof amino acids whose hydropathic indices are within ±2 is preferred,those within ±1 are particularly preferred, and those within ±0.5 areeven more particularly preferred.

Substitution of like amino acids can also be made on the basis ofhydrophilicity, particularly, where the biological functional equivalentpolypeptide or peptide thereby created is intended for use inimmunological embodiments. The following hydrophilicity values have beenassigned to amino acid residues: arginine (+3.0); lysine (+3.0);aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine(+0.2); glutamine (+0.2); glycine (0); proline (−0.5±1); threonine(−0.4); alanine (−0.5); histidine (−0.5); cysteine (−1.0); methionine(−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine(−2.3); phenylalanine (−2.5); tryptophan (−3.4). It is understood thatan amino acid can be substituted for another having a similarhydrophilicity value and still obtain a biologically equivalent, and inparticular, an immunologically equivalent polypeptide. In such changes,the substitution of amino acids whose hydrophilicity values are within±2 is preferred, those within ±1 are particularly preferred, and thosewithin ±0.5 are even more particularly preferred.

As outlined above, amino acid substitutions are generally based on therelative similarity of the amino acid side-chain substituents, forexample, their hydrophobicity, hydrophilicity, charge, size, and thelike. Exemplary substitutions that take various of the foregoingcharacteristics into consideration are well known to those of skill inthe art and include (original residue: exemplary substitution): (Ala:Gly, Ser), (Arg: Lys), (Asn: Gln, His), (Asp: Glu, Cys, Ser), (Gln:Asn), (Glu: Asp), (Gly: Ala), (His: Asn, Gln), (lie: Leu, Val), (Leu:Ile, Val), (Lys: Arg), (Met: Leu, Tyr), (Ser: Thr), (Thr: Ser), (Tip:Tyr), (Tyr: Trp, Phe), and (Val: lie, Leu). Embodiments of thisdisclosure thus contemplate functional or biological equivalents of apolypeptide as set forth above. In particular, embodiments of thepolypeptides can include variants having about 50%, 60%, 70%, 80%, 90%,and 95% sequence identity to the polypeptide of interest.

As used herein “functional variant” refers to a variant of a protein orpolypeptide (e.g., a circularly permuted protein, with or withoutadditional sequence alterations) that can perform the same functions oractivities as the original protein or polypeptide, although notnecessarily at the same level (e.g., the variant may have enhanced,reduced or changed functionality, so long as it retains the basicfunction).

“Identity,” as known in the art, is a relationship between two or morepolypeptide sequences, as determined by comparing the sequences. In theart, “identity” also refers to the degree of sequence relatednessbetween polypeptide as determined by the match between strings of suchsequences. “Identity” and “similarity” can be readily calculated byknown methods, including, but not limited to, those described in(Computational Molecular Biology, Lesk, A. M., Ed., Oxford UniversityPress, New York, 1988; Biocomputing: Informatics and Genome Projects,Smith, D. W, Ed., Academic Press, New York, 1993; Computer Analysis ofSequence Data, Part I, Griffin, A. M., and Griffin, H. G., Eds., HumanaPress, New Jersey, 1994; Sequence Analysis in Molecular Biology, vonHeinje, G., Academic Press, 1987; and Sequence Analysis Primer,Gribskov, M. and Devereux, J., Eds., M Stockton Press, New York, 1991;and Carillo, H., and Lipman, D., SIAM J Applied Math., 48: 1073 (1988).

Preferred methods to determine identity are designed to give the largestmatch between the sequences tested. Methods to determine identity andsimilarity are codified in publicly available computer programs. Thepercent identity between two sequences can be determined by usinganalysis software (e.g., Sequence Analysis Software Package of theGenetics Computer Group, Madison Wis.) that incorporates the Needelmanand Wunsch, (J. Mol. Biol., 48: 443-453, 1970) algorithm (e.g., NBLAST,and XBLAST). The default parameters are used to determine the identityfor the polypeptides of the present disclosure.

By way of example, a polypeptide sequence may be identical to thereference sequence, that is be 100% identical, or it may include up to acertain integer number of amino acid alterations as compared to thereference sequence such that the % identity is less than 100%. Suchalterations are selected from: at least one amino acid deletion,substitution, including conservative and non-conservative substitution,or insertion, and wherein said alterations may occur at the amino- orcarboxy-terminal positions of the reference polypeptide sequence oranywhere between those terminal positions, interspersed eitherindividually among the amino acids in the reference sequence or in oneor more contiguous groups within the reference sequence. The number ofamino acid alterations for a given % identity is determined bymultiplying the total number of amino acids in the reference polypeptideby the numerical percent of the respective percent identity (divided by100) and then subtracting that product from said total number of aminoacids in the reference polypeptide.

An “enzyme,” as used herein, is a polypeptide that acts as a catalyst,which facilitates and generally speeds the rate at which chemicalreactions proceed but does not alter the direction or nature of thereaction.

As used herein, the term “promoter” includes all sequences capable ofdriving transcription of a coding sequence. In particular, the term“promoter” as used herein refers to a DNA sequence generally describedas the 5′ region of a gene, located proximal to the start codon. Thetranscription of an adjacent gene(s) is initiated at the promoterregion. The term “promoter” also includes fragments of a promoter thatare functional in initiating transcription of the gene.

A “primer” as used herein generally refers to a nucleic acid strand, ora related molecule, that serves as a starting point for replication, andare used in amplification techniques, such as the polymerase chainreaction (PCR). Primers used in such techniques are usually relativelyshort (generally about 20-50 base pairs), artificially synthesizedpolynucleotide strands. In PCR, primers are used to select thepolynucleotide sequence to be amplified by the PCR process.

The term “expression” as used herein describes the process undergone bya structural gene to produce a polypeptide. It is a combination oftranscription and translation.

The term “plasmid” as used herein refers to a non-chromosomaldouble-stranded DNA sequence including an intact “replicon” such thatthe plasmid is replicated in a host cell.

As used herein, the term “vector” or “expression vector” is used inreference to a vehicle used to introduce a nucleic acid sequence into acell. A vector may include a DNA molecule, linear or circular, whichincludes a segment encoding a polypeptide of interest operably linked toadditional segments that provide for its transcription and translationupon introduction into a host cell or host cell organelles. Suchadditional segments may include promoter and terminator sequences, andmay also include one or more origins of replication, one or moreselectable markers, an enhancer, a polyadenylation signal, etc.Expression vectors are generally derived from yeast or bacterial genomicor plasmid DNA, or viral DNA, or may contain elements of both.

The term “transformation” refers to the introduction of DNA or RNA intocells in such a way as to allow gene expression.

The terms “native termini”, “original termini” or “native terminus”refer to the terminal amino acid residues of a protein prior to itscircular permutation (e.g., the amino and carboxy terminal ends of thenative or wild type protein).

The terms “new termini” or “new terminus” refer to the terminal aminoacid residues of a protein after its circular permutation. The “newtermini” or “new terminus” are different from the native or originaltermini.

The term “coupled” as used herein refers to the binding, bonding, orother forms of association of a protein, specifically the association ofa protein having an active site and a substrate or ligand.

As used herein, the term “enhance,” “increase,” and/or “augment”generally refers to the act of improving a function or behavior relativeto the natural, expected or average. For example, a circularly permutedprotein that has increased activity over that of the correspondingnative protein, has improved activity (e.g. a faster rate of reaction,or binding/reacting with a greater number of substrates in the sameamount of time) as compared to the activity of the corresponding nativeprotein.

The term “substantially similar” as used herein generally refers to afunction, activity, or behavior that is close enough to the natural,expected, or average, so as to be considered, for all practicalpurposes, interchangeable. For instance, a protein with substantiallysimilar activity would be one that has an activity level that would notbe considered to be substantially more or less active than the nativeprotein.

As used herein, the term “improvement” or “enhancement” generally refersto a change or alteration in a function or behavior of a protein, suchas an enzyme, that in the applicable circumstances is considered to bedesirable.

The term “accessibility” as used herein refers to the ability of asubstrate or ligand to associate with or couple the active site of aprotein/enzyme. Thus, a protein with “increased accessibility” is one inwhich substrates (including natural or novel substrates) are more easilyable to associate with or couple the active site of the protein, ascompared with the native or wild-type protein.

The term “enantioselectivity” as used herein refers to process forinteracting with a single desired enantiomer over others. Thus, aprotein with “increased enantio-selectivity” has a greater preferencefor one enantiomer over the other enantiomer, as compared to what isnatural or expected or to the native or another protein.

The term “substrate specificity” refers to the range of substrates thata polypeptide can act upon to produce a result. The term “broadersubstrate specificity” refers to a larger range of substrates that apolypeptide can act upon to produce a result, as compared to the nativeprotein. The term “changed substrate specificity” refers to a differentor altered range of substrates than a polypeptide can act upon toproduce a result, as compared to the native protein.

A residue or terminus that is “in or near” the active site of a proteinrefers to a residue or terminus that is sufficiently close to the activesite of the protein, when the protein is in its folded conformation, toaffect the accessibility, flexibility, and/or functionality of theactive site. The use of “in” or “near” are interchangeable.

The term “immobilized enzyme” refers to an enzyme bound covalently ornon-covalently to the surface of a solid or semi-solid surface material(e.g., a matrix material) including, but not limited to, ion-exchangebeads and agarose.

The term “reaction medium” refers to the environment in which the enzymeor immobilized enzyme catalyzes a chemical reaction. Typically, reactionmedium for lipases and esterases, for example, include, but are notlimited to, aqueous buffer solutions, organic solvents, and ionicliquids. Changes in the reaction medium are known to sometimes affectthe properties of an enzyme, altering, for example, its substratespecificity and enantio-selectivity. Additional adjustable parameters ofthe reaction medium include, but are not limited to, the water activityin non-aqueous reaction medium, as well as the nature of the reagents inthe chemical reaction including, but not limited to, vinyl acetate oracetic acid. In summary, optimization of the reaction medium for anenzyme-catalyzed reaction can be used to further improve the performanceof enzymes.

The term “conformation” in reference to a protein or peptide (e.g.“folded conformation”) generally refers to the higher folded states ofthe peptide beyond the primary structure (peptide sequence),particularly to the tertiary structure of the protein or peptide.

The term “secondary engineering” or “secondary mutation” refers to theact, or result thereof, of performing additional mutation, sequencealterations, or other protein engineering on a already mutated (e.g.,non-native, non wild-type) protein. For instance, a circularly permutedprotein already differs from the corresponding native protein in thelocation of its termini; thus, a secondary mutation of a circularlypermuted protein would include another mutation or variation (e.g. adeletion, substitution, or insertion) from the native protein, inaddition to the new termini location. Additional description andexamples of secondary engineering and secondary mutations are discussedin greater detail below.

Description:

The present disclosure generally provides compositions includingengineered proteins and peptides having increased activity and/or otherenhancements/improvements over the corresponding native or wild-typeproteins, where the amino-terminal and carboxy-terminal ends of theengineered proteins are relocated with respect to the amino-terminal andcarboxy-terminal ends of the native protein, as illustrated in FIG. 1.In other words, the present disclosure provides compositions includingactive, or functional, circularly permuted proteins having higher orenhanced activity and/or other improvements over the native protein(e.g., increased accessibility, increased active site flexibility,increased enantioselectivity, increased stability, and broader and/orchanged substrate specificity).

In one embodiment, the present disclosure provides circularly permutedproteins having the N and C-termini relocated to a location in or nearthe active site of the protein. As discussed further below, conventionalthought in the art of circular permutation for protein design dictatesthat the new N and C-termini of a circularly permuted protein should notgenerally be in a location near the active site and generally should notbe in a location known to form a part of an important secondarystructure or tertiary fold of the protein. This is due to valid concernsthat breaking the protein backbone at such a location could interferewith the folding and conformation, and thus the function, of theprotein, possibly to the extent of inhibiting all functionality.

However, the compositions and methods of the present disclosuredemonstrate that circularly permuted proteins having the terminirelocated to certain locations in or near the active site of a protein,not only do not destroy functionality, but can even enhancefunctionality of the protein, in some cases up to about 175-fold, overthe native protein. Moreover, the new amino-terminal andcarboxy-terminal ends of such enhanced-function, circularly permutedproteins may be located not just in external loop regions near theactive site of the protein, but also may be embedded in secondarystructures such as alpha helices, which are near or form a part of theactive site of a protein. In some embodiments the new amino-terminal andcarboxy-terminal ends of the circularly permuted proteins are locatedwithin about 20 Å from the active site of the circularly permutedprotein; in other embodiments the new termini are located within about15 Å from the active site of the circularly permuted protein.

In other embodiments, the present disclosure provides circularlypermuted proteins having the N and C-termini relocated to a location notin or near (e.g., distant from) the active site of the protein. Althoughthe new termini are not in or near the active site of the protein,preferably the new locus has a desirable effect on protein functionand/or behavior.

The present disclosure also provides libraries of circularly permutedproteins corresponding to a native protein of interest. The circularpermutation libraries of the present disclosure include one or morevariants of a protein of interest having relocated amino-terminal andcarboxy-terminal ends, where the relocated ends are in a differentlocation from the terminal ends of the native protein. Preferably, suchlibraries include circularly permuted variants having new terminal endsat locations throughout the polypeptide sequence. More preferably, suchlibraries include and can be screened for functional variants. Mostpreferably, such libraries include functional variants having increasedactivity or other improvements over the native protein. In someembodiments, the libraries include functional variants having newterminal ends at locations in or near the active site of the protein.

In some embodiments, the circularly permuted proteins of the presentdisclosure are proteins of the α/β-hydrolase fold family (e.g., lipases,esterases, acetylcholinesterase, dienelactone hydrolase, thioesterase,serine carboxypeptidase, proline iminopeptidase, proline oligopeptidase,haloalkane dehalogenase, haloperoxidase, epoxide hydrolase, andhydroxynitrile lyase). Many lipases and esterases have similarstructures and/or functions. As such, some references refer to somelipases as esterases and vice versa. It is the intent of this disclosureto include all proteins of the α/β-hydrolase fold family, some of whichmay be called lipases and esterases, but the exact term of lipase oresterase may be interchangeable in some embodiments (e.g., proteins fromCandida antarctica may be called lipases or esterases). Therefore,reference to lipase does not necessarily exclude esterase.

Lipases can catalyze the formation, hydrolysis, and substitution(transesterification) of ester bonds, amid bonds, and the like. They areimportant biocatalysts in production of chiral building blocks for finechemicals and pharmaceuticals, as well as in bulk products such aslaundry detergents. In particular in the context of kinetic resolutionand chiral synthesis, the enzymes' broad substrate specificity, theirhigh stability (e.g. tolerance of organic solvents and elevatedtemperatures), as well as their high enantio and regio-selectivity makesthem popular choices.

In preferred embodiments, the circularly permuted α/β-hydrolase foldfamily proteins or peptides (e.g., lipase) have increased activityand/or one or more other improvements, including but not limited to,increased stability, increased accessibility to the active site,increased active site flexibility, broader and/or changed substratespecificity, and/or increased enantioselectivity, as compared to thenative protein. In some embodiments the circularly permuted proteins ofthe α/β-hydrolase fold family have new terminal ends in or near theactive site. In some embodiments the new termini are located withinabout 20 Å from the active site of the circularly permuted protein; inother embodiments the new termini are located within about 15 Å from theactive site of the circularly permuted protein. In some preferredembodiments, the new terminal ends are located in the region known asthe “cap” domain or cap region of a α/β-hydrolase fold family protein.The cap domain generally refers to the region of the protein forming acap-like structure over the active site that may form part of the activesite binding pocket, but that does not generally form part of the coreα/β-hydrolase fold. FIG. 2 depicts two members of the α/β-hydrolase foldfamily, lipase B from Candida antarctica (CALB) (FIG. 2B) and theepoxide hydrolase from Agrobacterium radiobacter (FIG. 2C). As can beseen from the figure, both proteins contain the core α/β-hydrolase fold,a cap region, and the active site (with the three residues of thecatalytic triad) located generally between the core and the cap regions.In other embodiments, the circularly permuted α/β-hydrolase fold familyhave new terminal ends outside of or distant from the active site of theα/β-hydrolase fold family.

Although not intending to be bound by theory, in some embodiments of thepresent disclosure, the circularly permuted α/β-hydrolase fold familyprotein has broader and/or changed substrate specificity resulting fromincreased flexibility and/or accessibility of the active site allowingthe α/β-hydrolase fold family to couple or associate with substratesand/or ligands that it is normally unable to couple. Such substratesinclude, but are not limited to, amides, esters, and particularly estersof large secondary and tertiary alcohols.

The reaction medium represents another parameter in the performance ofindividual enzymes in biocatalysis. While the specific effects of theenvironment on the catalysts are, for the most part, poorly understood,the results from stochastic approaches clearly demonstrate that theoptimization of the reaction medium can affect the substrate specificityand enantioselectivity, as well as the protein stability. Reactionmedium engineering typically involves two aspects: a) the modificationof the enzyme catalyst itself, and b) the change of the reagent andsolvent environment. Although not intending to be bound by theory, inthe former, the enzyme can, for example, be used in its native form, bemodified by chemical reactions of (most likely) surface residues toimprove its solubility (for example nitration), or be immobilized onsolid or semi-solid support (e.g. a matrix material, such as beads, or acolumn). Although not intending to be bound by theory, in the lattercase, the choice of aqueous buffer solutions, organic solvents, andionic liquids and temperature not only affects the nature of thechemical reaction (hydrolysis versus esterification) but is known to beable to affect the properties of an enzyme, altering, for example, itsstability, substrate specificity and enantioselectivity. Additionaladjustable parameters of the reaction medium include, but are notlimited to, the water activity in non-aqueous reaction medium, as wellas the nature of the reagents in the chemical reaction including, butnot limited to, vinyl acetate or acetic acid. In summary, optimizationof the reaction medium for an enzyme-catalyzed reaction can be used tofurther improve the performance of enzymes.

Among the most commonly used biocatalysts in the lipase family is lipaseB from Candida antarctica (CALB) (ONA Sequence, SEQ ID NO: 1). CALB is a317 amino-acid protein (SEQ ID NO: 2) with the characteristicα/β-hydrolase fold as its core structure and the catalytic triadSer-His-Asp in the active site. A three dimensional representation ofCALB, illustrating the protein's secondary and tertiary structure, isshown in FIG. 6. CALB shows outstanding specificity and selectivity,especially for esters of secondary alcohols. Recent protein engineeringefforts have only added to the wide variety of reactions catalyzed bythis enzyme. Thus, embodiments of the present disclosure providecircular permutations of CALB. Certain embodiments provide that thecircularly permuted CALB has new amino- and/or carboxy terminal endslocated in α17, α16, α9, α7, or α2 (e.g., between residue 44 and residue47 of α2). Embodiments of the present disclosure include circularlypermuted CALB proteins having new amino-terminal ends in locationsincluding, but not limited to, residues 44, 144, 148, 150, 193, 268,277, 278, 283, 284, 289, and 294. Circularly permuted proteins will bedenoted herein by the prefix “cp-” and followed by the residue numberthat is the new amino terminus, for example, a circularly permutedprotein with the new N-terminus as residue 144 would be denoted ascp144. In some preferred embodiments, the new termini are located in thecap region of CALB (e.g., the region including α7, α9, α17, α19 and anyconnecting external loop regions). In a preferred embodiment, thecircularly permuted CALB has a new amino-terminal end located at residue283 (cp283).

The present disclosure also provides methods of using circularpermutation to design novel proteins, specifically enzymes, morespecifically members of the α/β-hydrolase fold family, most specificallylipases and esterases, with enhanced activity and/or one or more otherimprovements over the native protein including, but not limited to,increased stability, increased accessibility to the active site,increased active site flexibility, broader and/or changed substratespecificity, increased enantioselectivity or a combination thereof. Insome embodiments the improvement is due to increased flexibility and/oraccessibility added to the active site due to changing the location ofthe termini to a location in or near the active site of the protein. Itis also contemplated that changing the location of the termini to alocation distant from, or otherwise outside of, the active site of theprotein could also affect the conformational environment, or otheraspect, of the protein in such a way so as to result in one or more ofthe above improvements.

Briefly described, the methods of the present disclosure include, butare not limited to, selecting a native protein having an active site, anamino-terminal end and a carboxy-terminal end; linking theamino-terminal and carboxy-terminal ends of the native protein to form acircular protein molecule, preferably via a linker; creating a libraryof circularly permuted proteins having at least one, but preferablymultiple, circularly permuted protein in the library with a newamino-terminal end and carboxy-terminal end, which are different fromthe amino-terminal and carboxy-terminal ends of the native protein; andselecting functional variants from the library. The method may furtherinclude mapping the location of the new amino-terminal andcarboxy-terminal ends in the functional variants to determine locationsof permissible permutations and selecting functional variants withtermini in various different locations for further testing. Such furthertesting may include, but is not limited to, detailed kinetic analysis,enantioselectivity, substrate specificity, and structural analysis(e.g., via fluorescence spectroscopy, circular dichroism, and proteinengineering). Additionally, the methods of the present disclosure mayfurther include selecting, from the library of functional variants,circularly permuted proteins having amino-terminal and carboxy-terminalends located in or near a binding site of the protein, and thensubmitting such variants to further testing as described above. Thesemethods will be described in greater detail in the discussion andexamples below.

Using the methods of the present disclosure to introduce additionalflexibility to the protein, especially in the region of the active site,allows researchers to design proteins and peptides, especially enzymes,to have desired enhancements/improvements over the native protein.Examples of some possible enhancements include, but are not limited to,increased activity, increased accessibility, increasedenantioselectivity, increased stability, and broader and/or changedsubstrate specificity. It should be noted that these enhancements maynot be due or are only partially due to flexibility of the protein, andembodiments of the disclosure are not limited to this theory regardingflexibility.

In one embodiment of the method of the present disclosure, describedbriefly here and in greater detail in the examples below, a library ofengineered variants of CALB was generated by random circular permutationof the wild type protein. In several variants the relocation of theprotein's termini altered the biochemical and biophysical properties ofthe catalyst, resulting in novel and improved activity toward selectedsubstrates in response to changes in the active site geometry,substrate/product binding affinities, and/or protein flexibility.Functional variants among the library members were identified andsubjected to detailed studies of their biochemical and biophysicalproperties. These circularly permuted biocatalysts may find applicationsin kinetic resolutions, biotransformations, or as polymerizationcatalysts. Alternatively, these permutants can serve as templates forsecondary protein engineering approaches.

The present disclosure also includes methods of further engineering thecircularly permuted proteins of the present disclosure to produce asecond generation of circular permuted proteins (second circularlypermuted proteins) having secondary mutations (e.g. mutations and/oralterations resulting from secondary engineering efforts, in addition tothe alterations introduced by the initial circular permutation). Suchsecondary mutations include, but are not limited to, deletions,insertions, and substitutions of one or more amino acids in thepolypeptide sequence of the circularly permuted protein, andcombinations thereof. The secondary mutations result in one or moresecond circularly permuted proteins that preferably have at least oneimprovement as compared to the corresponding native protein and thecorresponding circularly permuted protein, which includes, but is notlimited to, increased activity, increased stability, increasedenantioselectivity, increased accessibility to the active site,increased active site flexibility, and broader and/or changed substratespecificity.

Secondary engineering approaches for introducing the secondary mutationsinclude, but are not limited to, various techniques of proteinengineering, such as mutations based on rational design and methods ofdirected evolution, such as insertion, deletion, or substitution of anindividual position or multiple positions in the protein sequence bymutagenesis, homology-dependent recombination, homology-independentrecombination, computational methods of directed evolution usingalgorithms (e.g., the SCHEMA algorithm). Secondary engineeringtechniques are known to those of skill in the art, and many of thetechniques listed above are described in Lutz, S., et al., “Novelmethods for directed evolution of enzymes: quality, not quantity,”(2004) Current Opinion in Biotechnology, 15:291-297, which is herebyincorporated by reference.

Exemplary secondary engineering efforts include, but are not limited to,rational and random mutagenesis, (as described in Cadwell, R. C. &Joyce, G. F. (1992) PCR methods and applications, 2, 28-33; andReidmann-Olsen, J. F. et al. (1991) Methods in Enzymology, 208, 564-586,which are hereby incorporated by reference), as well as in vitro and invivo recombination based on sequence homology. Examples of suchapproaches include, but are not limited to, DNA shuffling (as describedin Stemmer, W. P. (1994) Proc Natl Acad Sci USA, 91, 10747-10751;Stemmer, W. P. (1994) Nature, 370, 389-391; and Zhao, H., Giver, L.,Shao, Z., Affholter, J. A. and Arnold, F. H. (1998) Nat Biotechnol, 16,258-261, which are hereby incorporated by reference) and methods forengineering proteins independent of sequence homology (e.g., ITCHY &SCRATCHY and other methods as described in Ostermeier et al., “Acombinatorial approach to hybrid enzymes independent of DNA homology”(1999) Nature Biotechnology, 17: 1205-9; Lutz et al. “Creatingmultiple-crossover DNA libraries independent of sequence identity”(2001) Proc. Natl. Acad. Sci. USA 98:11248-53; and Sieber et al.“Libraries of hybrid proteins from distantly related sequences” (2001)Nature Biotechnology 19:456-60, which are hereby incorporated byreference).

In embodiments of the methods of the present disclosure, circularpermutation is performed on a protein of interest to generate a libraryof permutants with new termini. Then, functional variants are identifiedby screening for protein activity by methods known to those of skill inthe art, such as colony screening for enzyme activity, examples of whichare described in further detail in the examples below. The functionalvariants are then mapped to determine the locations of permissiblepermutations in the protein sequence that allow the protein to retainactivity. Then, representative functional permutants having new terminiat various locations in the protein sequence are chosen for furthertesting. In some embodiments, the representative permutants are testedfor detailed kinetic analysis to determine the relative activity withrespect to the native protein. This helps to identify permutants withincreased activity over that of the native protein.

The circularly permuted proteins can then also be tested for structuralintegrity via various methods known to those of skill in the artincluding, but not limited to, fluorescence spectroscopy and circulardichroism, both of which are described in greater detail in the examplesbelow. Structural analysis of the protein helps to determine whateffects the new location of the termini have on the local or overallstructure of the protein. This can help identify proteins that havegreater accessibility to the active site and/or greater active siteflexibility, which may explain a higher level of activity. Structuralanalysis can also help to identify possible targets for secondaryengineering efforts, such as by identifying areas of the protein thatmay lead to structural instability.

The circularly permuted proteins can also be tested forenantioselectivity to determine if they retain or have improvedenantioselectivity over the native protein. In preferred embodiments,the circularly permuted protein(s) will have at least substantiallysimilar enantioselectivity to the native protein. Various permutants mayalso be tested to determine how circular permutation affects thespecificity, selectivity, and promiscuity of the protein. For instance,tests can be performed to measure the kinetic properties of functionalvariants on various selected substrates. Preferably, the circularpermutants are tested on substrates from three categories: 1) naturalsubstrates to probe for retention of wild type specificity andselectivity, 2) unnatural substrates to test for novel activity, and 3)on substrates no typically associated with the particular type ofprotein or enzyme to investigate whether circular permutation can giverise to promiscuous activity.

In some embodiments, the permutants, or those of particular interest,are tested for stability, since stability is a factor in the performanceof the protein in certain environments that might be relevant forpossible commercial use. In some embodiments, the circularly permutedproteins are also coupled to a surface/substrate, such as a matrix, forsome or all of the above testing. Such substrates are known to those ofskill in the art, and some exemplary substrates are described in theexamples below.

In one non-limiting embodiment of the present disclosure, described ingreater detail below, lipase B from Candida antarctica (CALB) wascircularly permuted and various circular permutants were subject tofurther analysis and testing as described above. Additionally, acircular permutant of particular interest was identified and subject tosecondary engineering techniques to generate a library of secondarycircularly permuted proteins containing secondary mutations. Thesesecondary permutants were then tested for various functions andbehaviors according to the methods of the present disclosure. Details ofthis exemplary embodiment of the disclosure are described in detailbelow along with a detailed discussion of circular permutationtechniques.

The introduction of the new and powerful combinatorial proteinengineering methods of this disclosure provide the ability to acceleratethe discovery of tailored catalysts for specific, synthetic problems andenvironmental constraints, giving the methods of this disclosure thepossibility to play a dominant role in the future of proteinengineering.

Circular Permutation:

Circular permutation is a little-explored technique for thediversification of protein frameworks useful in designing new and/orimproved proteins and peptides. As discussed in more detail below andillustrated in FIG. 1A, circular permutation involves the connection ofa protein's 10 natural termini 12 and 14 by a linker 26, preferably apeptide linker, followed by the reintroduction of new termini 22 and 24in another region of the protein framework to produce one or morecircular permutants 20. The termini relocation may affect the structuralintegrity of the protein, changing its active site accessibility andflexibility, all factors affecting an enzyme's substrate recognition andturnover. While surface loop regions seem preferred choices for newends, experimental studies, as described in the examples below, havedemonstrated that termini in secondary structures and the core region ofa protein are also possible. In one embodiment of the disclosure, acomplete combinatorial library of circular permuted CALBs (FIG. 3) wasgenerated in order to maximize the efficiency and information content ofthe experiments.

Circularly permuted proteins have been found naturally in variousorganisms, including viruses, bacteria, plants, and higher animals. Theyare derived from either posttranslational modification, gene duplicationor from exon shuffling events. Concanavalin A, a circularly permutedform of favin, was the first reported permuted protein in eukaryotesformed by post-translational transposition and ligation within theinitial polypeptide. Swaposin, which is a plant aspartic proteinaseinsert, is the circularly permuted form of saposin. In 1995 Russell andcoworkers found that although swaposin is highly homologous to saposinwith four helices and a disulphide bond in structure, the two N-terminalhelices of saposin are swapped to the C-terminal in swaposin andconnected by a polypeptide linker. cDNA analysis of swaposin revealedthat circular permutation occurs on the gene level instead of throughposttranslational modification. Circular permutation of natural proteinsmay be of functional importance. In case of swaposin, it washypothesized that the movement of the termini may facilitate theinsertion of the swaposin domain within the aspartic proteinase, takingadvantage of the orientation difference between swaposin and originalsaposin domain.

Another example of circular permutation is the aldolase superfamily.Members of this superfamily share a common TIM barrel fold, whichcontains eight α/β motifs assembled in a circular arrangement. Thisstructural character may assist the occurrence of circular permutation,and enzymes with high similarity in substrate specificities and reactionchemistries except for different active site locations were revealed. Itis proposed that the active site flexibility may account in part for thefurther adaptation for new functions, which possibly gives anexplanation to the functional diversity of the TIM barrel in nature.

In the laboratory, circular permutation was first carried out on bovinepancreatic trypsin inhibitor through chemical condensation. In 1989 theuse of genetic engineering was first used to design circularly permutedanthranilate isomerase. The termini relocation may afford valuableinformation about the importance of the natural ends of the polypeptidechain in respect to tertiary structure and biological function. It isbelieved that critical structure elements can not be disrupted by abreakage in the backbone, while chain connectivity is believed to affectthe transition state and the folding nucleus of a protein. An example ofthe impact of circular permutation on protein function is the fusionprotein between interleukin 4 and exotoxin from Pseudomonas, where thesimple back-to-back fusion of the two components deactivated theinterleukin but function was restored upon reorganization of the fusionprotein by circular permutation.

Compared to rational design approaches, random circular permutationprovides a more comprehensive approach to study protein stability andthe relationship between protein structure and catalysis. Rather thangenerating one permutation per experiment, a complete set of allpossible termini relocations are generated in a single test tube andevaluated by high-throughput screening or selection methods. Thismethodology can be applied to numerous and varied proteins, and inparticular to enzymes, to engineer proteins with improved function overtheir native counterparts. In an embodiment of the present disclosure,circular permutation was applied to the exploration of CALB's structuraland functional diversity.

It will be appreciated that while circular permutation is described interms of linking the two ends of a protein and then cutting thecircularized protein, these steps are not actually required to createthe end product. Thus, circularized permutations of a generic proteinwith any of the novel sequences disclosed herein refers to all proteinsof such structure regardless of how they are constructed.

It is important to create a permutation that will retain the biologicalactivity of the native form of the molecule. If the new terminiinterrupt a critical region of the native protein, activity may be lost.Similarly, if linking the original termini destroys activity, it islikely that no permutation will retain native biological activity. Thus,there are two preferred, but limiting, attributes of a candidate for thecreation of an active circularly permuted protein: 1) termini in thenative protein that are favorably located so that creation of a linkagedoes not destroy native biological activity; and 2) an “opening site”that exists where new termini can be formed without functionallydisrupting a region critical for protein folding and desired biologicalactivity.

Thus, in general, good candidates for circular permutation are proteinsin which the termini of the original protein are in close proximity andfavorably oriented. Where the termini are naturally situated closetogether, it is expected that direct fusion of the termini to each otheror introduction of a linker will have relatively little effect. It hasbeen suggested that in roughly one third of the known structures ofglobular proteins the termini are in relatively close proximity (Thortonet al. J. Mol. Biol., 167: 443-460 (1983)). However, because the linkermay be of any length, close proximity of the native termini is not anabsolute requirement.

In a preferred embodiment, it is desirable to use a linker thatpreserves the spacing between the termini comparable to the unpermutedor native molecule. Generally, linkers are either hetero- orhomo-bifunctional molecules that contain two reactive sites that mayeach form a covalent bond with the carboxyl and the amino terminal aminoacids respectively. Suitable linkers are well known to those of skill inthe art and include, but are not limited to, straight or branched-chaincarbon linkers, heterocyclic carbon linkers, or peptide linkers. Themost common and simple example is a peptide linker that typicallyincludes several amino acids joined through peptide bonds to the terminiof the native protein. The linkers may be joined to the terminal aminoacids through their side groups (e.g., through a disulfide linkage tocysteine). However, in a preferred embodiment, the linkers will bejoined through peptide bonds to the alpha carbon amino and carboxylgroups of the terminal amino acids. In addition, direct linking of thenative protein termini via a peptide bond is possible in some proteins.

Functional groups capable of forming covalent bonds with the amino andcarboxyl terminal amino acids are well known to those of skill in theart. For example, functional groups capable of binding the terminalamino group include anhydrides, carbodimides, acid chlorides, activatedesters, amides, and the like. Similarly, functional groups capable offorming covalent linkages with the terminal carboxyl include amines,alcohols, and the like. In a preferred embodiment, the linker willitself be a peptide and will be joined to the protein termini by peptidebonds.

Conventional thought indicates that circular permutation requires thatthe protein have an opening site where the formation of termini will notinterrupt secondary structure crucial in the folding process or criticalelements of the final conformation. This is based on the belief that,even if the three-dimensional structure is compatible with joining thetermini, it is conceivable that the kinetics and thermodynamics offolding would be greatly altered by circular permutation if opening thecircularized protein separates residues that participate in short rangeinteractions crucial for the folding mechanism or the stability of thenative state. Goldenberg, Protein Eng., 7: 493-495 (1989). Thus, currentpractice advises that opening sites be selected in regions of theprotein that do not show secondary structure such as alpha helices,pleated sheets, barrel structures, and the like.

While it is true that the choice of an opening site is important to theprotein activity, it is not always the case that the new termini cannotbe located within secondary structure elements or near the active siteof the protein without negatively affecting the function of the protein.In fact, the compositions of this disclosure preferably include proteinswhere the new termini are located in or near the active site, whilestill preserving or even enhancing, the activity of the protein, inorder to confer greater flexibility or other desirable characteristicsto the active site and the circularly permuted protein as a whole. Insome preferred embodiments, the new termini are located within about 20Å from the active site of the circularly permuted protein; in otherembodiments the new termini are located within about 15 Å from theactive site of the circularly permuted protein. In some embodiments, thenew termini are located between about 5 Å and 20 Å of the active site,between about 5 Å and 15 Å, or between about 10 Å and 15 Å of the activesite.

Circularly permuted proteins may be made by a number of methods known tothose of skill in the art. These include chemical synthesis,modification of existing proteins, and expression of circularly permutedproteins using recombinant DNA methodology.

Where the protein is relatively short (e.g., less than about 50 aminoacids) the circularly permuted protein may be synthesized using standardchemical peptide synthesis techniques. If the linker is a peptide it maybe incorporated during the synthesis. If the linker is not a peptide, itmay be coupled to the peptide after synthesis. Solid phase synthesis inwhich the C-terminal amino acid of the sequence is attached to aninsoluble support followed by sequential addition of the remaining aminoacids in the sequence is one method for the chemical synthesis ofcircularly permuted proteins. Techniques for solid phase synthesis aredescribed by Barany and Merrifield, Solid-Phase Peptide Synthesis; pp.3-284 in The Peptides: Analysis, Synthesis, Biology. Vol. 2: SpecialMethods in Peptide Synthesis, Part A., Merrifield, et al. J. Am. Chem.Soc., 85: 2149-2156 (1963), and Stewart et al., Solid Phase PeptideSynthesis, 2nd ed. Pierce Chem. Co., Rockford, Ill. (1984), which areincorporated herein by reference.

Alternatively, the circularly permuted protein may be made by chemicallymodifying a native protein. Generally, this includes reacting the nativeprotein in the presence of the linker to form covalent bonds between thelinker and the carboxyl and amino termini of the protein, thus forming acircular protein. New termini are then formed by opening the peptidebond and then joining the amino acids at another location. This may beaccomplished chemically or enzymatically using, for example, apeptidase.

If the opening reaction tends to hydrolyze more than one peptide bond,the reaction may be run briefly. Those molecules having more than onepeptide bond opened will be shorter than the full length circularlypermuted molecule, and the latter may be isolated by any proteinpurification technique that selects by size (e.g., by size exclusionchromatography or electrophoresis). Alternatively, various sites in thecircular protein may be protected from hydrolysis by chemicalmodification of the amino acid side chains, which may interfere withenzyme binding, or by chemical blocking of the vulnerable groupsparticipating in the peptide bond.

In a preferred embodiment, circularly permuted proteins can besynthesized using recombinant DNA methodology, as illustrated in FIG.1B. Generally this involves creating a DNA sequence 30 that encodes thecircularly permuted protein 32 (including an original/native N-terminus34 and C-terminus 36), and DNA sequences 38 a and 38 b encoding for thelinker 38. The DNA sequence 30 is then circularized by intramolecularDNA ligation. The circularized DNA 40 is then cut and linearized byDNaseI. In preferred embodiments, the amount of DNaseI is minimized inorder to achieve generally only one cut per DNA sequence. Cutting andlinearization of the circular DNA sequences 40 produces one or morecircularly permuted DNA sequences 50 having new ends 54 and 56, encodingnew amino and carboxy termini, respectively, of the encoded circularlypermuted protein. The resulting circularly permuted proteins can beexpressed by placing the circularly permuted DNA sequences 50 in anexpression cassette under the control of a particular promoter,expressing the protein in a host, isolating the expressed protein and,if appropriate, renaturing the protein.

DNA encoding circularly permuted proteins may be prepared by anysuitable method, including, for example, cloning and restriction ofappropriate sequences or direct chemical synthesis by methods such asthe phosphotriester method of Narang et al. Meth. Enzymol. 68: 90-99(1979); the phosphodiester method of Brown et al., Meth. Enzymol. 68:109-151 (1979); the diethylphosphoramidite method of Beaucage et al.,Tetra. Lett., 22: 1859-1862 (1981); and the solid support method of U.S.Pat. No. 4,458,066, all incorporated herein by reference.

Chemical synthesis produces a single stranded oligonucleotide. This maybe converted into double stranded DNA by hybridization with acomplementary sequence, or by polymerization with a DNA polymerase usingthe single strand as a template. One of skill would recognize that whilechemical synthesis of DNA is limited to sequences of about 100 bases,longer sequences may be obtained by the ligation of shorter sequences.Alternatively, subsequences may be cloned and the appropriatesubsequences cleaved using appropriate restriction enzymes. Thefragments may then be ligated to produce the desired DNA sequence.

In a preferred embodiment, DNA encoding the circularly permuted proteinmay be produced using DNA amplification methods, for example polymerasechain reaction (PCR). First, the segments of the native DNA on eitherside of the new terminus are amplified separately. For example, sincethe native protein sequence of CALB is 317 amino acids long and theopening site is between amino acids 37 and 38 respectively, thesequences representing codons 1 through 37 and 38 through 317 areamplified separately. The 5′ end of the first amplified sequence encodesthe peptide linker, while the 3′ end of the second amplified sequencealso encodes the peptide linker. Since the 5′ end of the first fragmentis complementary to the 3′ end of the second fragment, the two fragments(after partial purification, e.g., on LMP agarose) can be used as anoverlapping template in a third PCR reaction. The amplified sequencewill contain codons 38-317, the linker, and codons 1-37. The circularlypermuted molecule may then be ligated into a plasmid.

The circularly permuted proteins may be expressed in a variety of hostcells, including, but not limited to, E. coli, other bacterial hosts,Pichia pastoris, Saccharomyces cerevisia, other yeast or fungi, andvarious higher eukaryotic cells such as the COS, CHO and HeLa cellslines and myeloma cell lines. The recombinant protein gene will beoperably linked to appropriate expression control sequences for eachhost. For E. coli this includes a promoter such as the T7, trp, orlambda promoters, a ribosome binding site and preferably a transcriptiontermination signal. For eukaryotic cells, the control sequences willinclude a promoter and preferably an enhancer derived fromimmunoglobulin genes, SV40, cytomegalovirus, etc., and a polyadenylationsequence, and may include splice donor and acceptor sequences.

The plasmids of the disclosure can be transferred into the chosen hostcell by well-known methods such as electroporation or calcium chloridetransformation for E. coli and calcium phosphate treatment orelectroporation for mammalian cells. Cells transformed by the plasmidscan be selected by resistance to antibiotics conferred by genescontained on the plasmids, such as the amp, gpt, neo and hyg genes.

Once expressed, the recombinant proteins can be purified according tostandard procedures of the art, including ammonium sulfateprecipitation, affinity columns, column chromatography, gelelectrophoresis and the like (see, generally, R. Scopes, ProteinPurification, Springer-Verlag, New York (1982), Deutscher, Methods inEnzymology Vol. 182: Guide to Protein Purification, Academic Press, Inc.New York (1990)). Substantially pure compositions of at least about 90to 95% homogeneity are preferred, and 98 to 99% or more homogeneity aremost preferred for applications. Once purified, partially or tohomogeneity as desired, the polypeptides may then be used in any desiredapplication.

One of skill in the art would recognize that after chemical synthesis,biological expression, or purification, the circularly permuted proteinmay possess a conformation substantially different than the nativeprotein. In this case, it may be appropriate to denature and reduce theprotein and then to cause the protein to re-fold into the preferredconformation. Methods of reducing and denaturing the protein andinducing re-folding are well known to those of skill in the art. (See,Debinski et al. J. Biol. Chem., 268: 14065-14070 (1993); Kreitman andPastan, Bioconjug. Chem., 4: 581-585 (1993); and Buchner, et al., Anal.Biochem, 205: 263-270 (1992), which are incorporated herein byreference.) Debinski et al., for example, describe the denaturation andreduction of inclusion body proteins in guanidine-DTE. The protein isthen refolded in a redox buffer containing oxidized glutathione andL-arginine.

One of skill would recognize that modifications could be made to thecircularized protein without diminishing its biological activity. Somemodifications may be made to facilitate the cloning, expression, orincorporation of the circularly permuted ligand into a fusion protein.Such modifications are well known to those of skill in the art andinclude, for example, a methionine added at the amino terminus toprovide an initiation site, or additional amino acids placed on eitherterminus to create conveniently located restriction sites or terminationcodons. For example, in some embodiments, circularly permuted proteinswill have an additional methionine (Met) at the amino terminus toprovide an initiation site. Circularly permuted proteins may alsocontain additional elements for cloning purposes.

One of skill will recognize that other modifications may be made. Thus,for example, amino acid substitutions may be made that increasespecificity or binding affinity of the circularly permuted protein, etc.Alternatively, non-essential regions of the molecule may be shortened oreliminated entirely. Thus, where there are regions of the molecule thatare not themselves involved in the activity of the molecule, they may beeliminated or replaced with shorter segments that merely serve tomaintain the correct spatial relationships between the active componentsof the molecule.

Design of Lipases:

The following describes some non-limiting examples of the presentdisclosure. It should also be noted that although scientific assertionsare made regarding how and/or why certain observations occur, there isno intent to be limited to these scientific assertions or to be bound bytheory.

In some exemplary embodiments of the present disclosure, circularpermutation was used to explore the effects of altered active siteaccessibility and protein backbone flexibility on the catalyticperformance of lipase B from Candida antarctica (CALB). CALB was chosenin part because it is a member of the α/β-hydrolase fold family, and itswide use as a biocatalyst in applications in biotechnology and organicsynthetic chemistry.

The α/β-hydrolase fold is one of the most versatile and widespreadprotein architectures and includes functionally diverse enzymes such asesterases, proteases, lipases, dehalogenases, haloperoxidases, lyases,and epoxide hydrolases. The structures of two members of theα/β-hydrolase fold family are illustrated in FIG. 2. Giving the fold itsname, the common feature in these enzymes is a conserved eight-strandedmostly parallel α/β-structure (FIG. 2A) which arranges in a twistedβ-sheet, flanked on both sides by

helices (FIGS. 2B and 2C). The α/β-hydrolase fold, or core, provides astable scaffold for the catalytic residues, typically a highly conservedtriad. Beyond the conserved core structure, members of this fold showtheir evolutionary potential by accommodating a wide variety of loopinsertions. Located mainly in the C-terminal half of the protein, theseinsertions can range from a few amino acids to entire domains, forminglids and caps that serve important roles by defining thesubstrate-binding pocket, and regulating accessibility of the activesite.

A number of enzymes in this fold family play an important role asbiocatalysts for asymmetric synthesis. Their broad substrate specificityand generally high regio and enantioselectivity makes the enzymesversatile tools for organic synthetic chemistry and biotechnology.Significant protein engineering efforts have been undertaken tocustomize these biocatalysts. Practitioners have adjusted the enzymes'thermostability and performance in organic solvents, as well as alteredthe substrate specificity and changed the enantioselectivity viarational design and directed evolution methods, but circular permutationhas not been used with this family of proteins to engineer theseenzymes.

CALB, a 317 amino acid-long enzyme, includes the α/β-hydrolase corestructure, which includes the residues of the catalytic triad (S105,D187, H224), and an extended cap domain near the protein's C-terminus.CALB shows outstanding biocatalytic characteristics for thestereoselective conversion of primary and secondary alcohols and is awidely used biotransformation catalyst.

Construction of circular permutation libraries and identification offunctional variants: Rather than substituting amino acids, it isbelieved that structural constraints in lipases could be relaxed throughprotein backbone cleavage. Specifically, it is believed that theinternal relocation of a protein's N and C-termini in or near the activesite can increase chain flexibility and active site accessibility, whichcould translate into higher activity for structurally more demandingsubstrates. Thus circular permutation was employed to explore theeffects of termini relocation on a lipase's catalytic performance.

Using a combinatorial approach, circular permutation of CALB identified63 unique functional protein permutants, and kinetic analysis ofselected candidates indicated that a majority of enzyme variants eitherretained or surpassed wild type CALB activity on a series of standardsubstrates. Beyond the potential benefits of these tailor-made lipasesas new catalysts for unnatural substrates, these results validatecircular permutation as a promising general method for proteinengineering, and in particular lipase engineering.

Given the difficulty of identifying suitable permutation sites byrational design, a comprehensive, combinatorial library of randomlypermuted CALB variants was generated. Starting with wild type CALB gene,flanking oligonucleotide sequences were first introduced which encodefor the flexible six-amino acid linker (-GGTSGG-) (SEQ ID NO: 3) tobridge the ˜17 Å distance between the original termini. Afterintramolecular ligation, the circular DNA was linearized in randompositions using DNaseI, as generally illustrated in FIG. 1B. Suchmethods are known to those of skill in the art and are described in thefollowing, which are hereby incorporated by reference in their entirety:Baird, G. S., et al., Proc. Natl. Acad. Sci. U.S.A. 1999, 96, (20),11241-11246; Beernink, P. T., et al., Protein Sci. 2001, 10, (3),528-537; and Graf, R., et al., Proc. Natl. Acad. Sci. U.S.A. 1996, 93,(21), 11591-11596. Reaction conditions were chosen such that, onaverage, only a single cut per DNA strand was introduced.

The resulting library of CALB permutants was then cloned into pPIC9 andtransformed into Pichia pastoris GS115 for protein expression asdescribed in greater detail below. DNA sequence analysis of 96 randomlychosen members in the naïve library (˜5×10⁵ colonies) confirmed theunbiased distribution of new termini over the entire length of theprotein sequence, which is illustrated by the circular permutation mapof CALB in FIG. 3. Next, functional variants in the CALB library wereidentified by colony screening on tributyrin plates as shown in FIG. 4and described in greater detail in the examples below. The DNA sequenceanalysis of functional members identified 63 unique protein sequenceswith termini in positions other than wild type, which are shown as thelines in the outer circle on the circular permutation map of CALB shownin FIG. 5.

The data indicate that CALB tolerates permutations in numerous positionsover the entire length of the protein. When mapped on the wild type CALBstructure, the new termini of functional permutants coincide not onlywith surface loops but interrupt secondary structure elements on theenzyme's surface and interior regions as shown by the patterned regionsin FIG. 6. Most noticeable is the concentration of functionalpermutations from amino acid 243 to 317, which make up the main portionof the enzyme's cap domain. This sequence is largely surface-exposed,wrapping around the front of the α/β-hydrolase core and forming thealcohol-binding portion of the active site pocket (α17).

Two additional regions tolerant to permutation include, but are notlimited to amino acids 44 and 47, which are located in close proximityto the oxyanion-stabilizing residues and a cluster of permutations inα7/9 (amino acid 135 to 155). This second region constitutes theenzyme's lid region and is also a part of the cap domain. Two proteinsegments (residues 48-143 and 204-246) were identified with nofunctional permutation. These regions make up the core of theα/β-hydrolase fold and include residues S105 and H224 of the catalytictriad. It is believed that the absence of functional permutation nearthese residues, as well as the presence of only a single site proximalto the triad's third amino acid (D187) reflects this region's importanceto catalysis and possibly its relevance to protein folding.

Kinetic analysis of protein variants: To examine the impact of circularpermutation on catalysis eleven functional CALB variants with termini inor near the active site were selected for detailed kineticcharacterization. The locations of the termini in these variants areshown on the structure of CALB in FIG. 6; the numbers correspond to theamino acid residue of the new terminus. Following overexpression in P.pastoris, the selected circularly permuted proteins were purified tohomogeneity. The catalytic performance of these variants was determinedin activity assays with two standard lipase substrates, measuring theinitial rates of hydrolysis of the chromogenic substrate p-nitrophenolbutyrate (pNB) and the fluorogenic substrate6,8-difluoro-4-methylumbelliferyl (DiFMU) octanoate. The kinetic dataare shown in Tables 1 and 2 below.

TABLE 1 Kinetic constants for CALB variants with p-nitrophenol butyrate(pNB). k_(cat) k_(cat) relative enzyme^(a) sequence^(b) K_(M)(μM)(min⁻¹) (min⁻¹ μM⁻¹) specificity^(c) wild type L1/P317 410 ± 40 305 ± 100.74 1.0 cp44 G44/T43 690 ± 90   6 ± 0.5 0.01 0.01 cp144 His-L144/A141550 ± 50 178 ± 7  0.32 0.4 cp144a L144/A141  820 ± 100 435 ± 28 0.53 0.7cp148 His-A148/L147 500 ± 30 171 ± 4  0.34 0.5 cp148a A148/L147 550 ± 70481 ± 29 0.87 1.2 cp150 His-S150/V149 510 ± 90 520 ± 45 1.02 1.4 cp150S150/V149 425 ± 50 347 ± 16 0.82 1.1 cp268 P268/T267 580 ± 90 3051 ± 2295.26 7.1 cp277 L277/A276  820 ± 100 1356 ± 94  1.65 2.2 cp278 L278/L2771180 ± 160 3117 ± 269 2.64 3.6 cp283 His-A283/A283- 280 ± 50 2971 ± 18010.61 14.3 KRPRINSP cp283a A283/A282 410 ± 60 3251 ± 206 7.93 10.7 cp284His-A284/A287- 550 ± 70 2980 ± 200 5.42 7.3 KRPRINSP cp284a A284/A283520 ± 80 4380 ± 298 8.42 11.4 cp289 His-P289/A284- 260 ± 30 3258 ± 21512.53 16.9 KRPRINSP cp289a P289/A288 790 ± 85 8055 ± 455 10.17 13.7cp294 His-E294/A283 310 ± 40 73 ± 4 0.23 0.3

TABLE 2 Kinetic constants for CALB variants with6,8-difluoro-4-methylumbelliferyl (DiFMU) octanoate. k_(cat) k_(cat)relative enzyme^(a) sequence^(b) K_(M)(μM) (min⁻¹) (min⁻¹ μM⁻¹)specificity^(c) wild type L1/P317 2.6 ± 0.3   2 ± 0.1 0.8 1.0 cp44G44/T43 5.6 ± 0.8  0.5 ± 0.05 0.1 0.13 cp144 His-L144/A141 2.0 ± 0.5   1± 0.1 0.5 0.6 cp144a L144/A141 3.0 ± 0.3 1.5 ± 0.1 0.5 0.6 cp148His-A148/L147 3.5 ± 0.5 1.5 ± 0.2 0.35 0.4 cp148a A148/L147 6.1 ± 0.73.5 ± 0.2 0.57 0.7 cp150 His-S150A/149 2.7 ± 0.8 2.1 ± 0.2 0.8 1.0 cp150S150/V149 5.5 ± 0.9 2.2 ± 0.2 0.4 0.5 cp268 P268/T267 2.3 ± 0.3 28.8 ±1.1  12.5 15.6 cp277 L277/A276 3.4 ± 0.5 16.3 ± 0.9  4.9 6.1 cp278L278/L277 5.2 ± 0.8 49.9 ± 3.2  9.7 12.1 cp283 His-A283/A283- 2.5 ± 0.5 25 ± 1.4 10.9 13.6 KRPRINSP cp283a A283/A282 2.4 ± 0.4 340 ± 17  140175 cp284a A284/A283 2.2 ± 0.2 242 ± 8  112 140 cp289 His-P289/A284- 5.5± 1.0 120 ± 7  23 28.8 KRPRINSP cp289a P289/A288 5.1 ± 1.0 150 ± 13  3037.5 cp294 His-E294/A283 9.5 ± 2.0  2.6 ± 0.34 0.3 0.4 In Tables 1 and2: ^(a)CALB nomenclature, e.g. cp44, indicates a circularly permutedprotein whose N-terminus starts at amino acid 44 of the wild typesequence; an “a” after the name indicates a variation of the particularcp-variant where tags and certain engineering artifacts (e.g., His tags,or C-terminal extensions) have been removed. The sequence^(b) indicatesthe N and C-terminal amino acids (all in single-letter code); smallvariations in chain length of individual permutants are caused byreading frame shifts and staggered ends upon DNasel digestion; Hisindicates the presence of a His tag, and additional sequence fragmentsare also indicated by single letter code. ^(c)Relative specificity =k_(cat)/K_(M) (variant)/k_(cat)/K_(M) (wild type).

The kinetic analysis confirmed that circular permutation has asignificant impact on CALB's catalytic performance. The most substantialimprovements in enzymatic activity over wild type CALB were observedupon termini relocation into the cap region of α16/17. Six of the sevenvariants (cp268-cp294) showed consistent improvements in their apparentk_(cat)'s for pNB and DiFMU octanoate. Three of the four variants(cp283, cp 284, cp289) show a consistent 10-fold improvement in theirapparent k_(cat)'s for pNB and up to 175-fold increases in DiFMUoctanoate turnover. Removal of the C-terminal peptide extension, anengineering artifact found in all three variants, and the His tag leftcatalytic rates generally unchanged or improved them, with a significantimprovement in the case of cp283. In contrast, a removal of the entireprotein fragment (amino acids 284-293) in cp294 appears detrimental tocatalysis. Whether the deletion dismantles the active site pocket,preventing productive substrate binding, or affects protein stability asthe disulfide-bond forming C293 is eliminated remains unclear.

The backbone cleavage in the lid region (cp144, cp148, cp150) showedmoderate effects on hydrolysis of our test substrates. Both K_(M) andk_(cat) for all three variants stay within two-fold of the parent enzymeunder the described assay conditions. Structure models predict closeinteractions of this protein region with the substrate's acyl portion.Furthermore, circular permutation of the lid region may alter theenzyme's response to changes in the reaction medium. The latter canaffect lipase activity by modulating conformational changes in the lidregion.

Finally, the kinetic data for cp44 shows a 10 to 100-fold reduction inrelative specificity, compared to wild type CALB. The close proximity ofthe permutation site to the oxyanion-binding pocket likely results inthe topological misalignment of the active site residues. Consistentwith the observation of permutation-free protein segments, it isbelieved that protein permutation does increase local backboneflexibility. While such flexibility seems detrimental at positions inproximity to active site residues, the relaxation effects can bebeneficial when applied to protein regions, which contribute to theactive site topology but do not directly carry a side chain involved incatalysis.

In summary, CALB engineering by circular permutation has generated atleast 63 new, unnatural lipase variants. Kinetic analysis confirmed thatthese protein variants can have sustained or improved catalytic functionon multiple substrates over wild type, mutant, and shuffled CALBs. Theobserved rate enhancements are believed to result from improved activesite accessibility and increased local protein backbone flexibility.

Analysis of substrate specificity and enantioselectivity in proteinvariants: To assess the circularly permuted CALB variants, three naturalsubstrates were selected to probe for retention of wild type specificityand selectivity (e.g., compounds 1-3 below).

Three natural substrates for CALB, (1) 4-cyclopentene-1,3-diol 1; (2)3-hydroxy-tetrahydrofuran; and (3) 6-methyl-5-hepten-2-ol, were testedas substrates for wild type CALB and cp283. The pure isomers of allthree compounds are important chiral building blocks in organicsynthetic chemistry, serving as starting materials for numerouspharmaceuticals. Additional compounds were also tested, and thesestudies are described in detail in Example 12 below and FIG. 11.

The experiments are typically performed in organic solvent, usingimmobilized enzymes and vinyl acetate as the second reagent, asdescribed in Wang Y F, et. al, Lipase-Catalyzed IrreversibleTransesterifications Using Enol Esters as Acylating Reagents—PreparativeEnantioselective and Regioselective Syntheses of Alcohols, GlycerolDerivatives, Sugars, and Organometallics. J Am Chem Soc 1988,110:7200-7205, which is hereby incorporated by reference in itsentirety. This reaction scheme has the advantage that the vinyl alcoholside product quickly rearranges into formaldehyde, effectively removingit from the reaction equilibrium and thereby avoiding productinhibition. Conversely, for reactions in organic solvents the enzyme ispreferably immobilized. The CALB variants were immobilized on LewatitVPOC 1600, a weak ion-exchange resin also used for commercial CALBproducts. In preliminary experiments, all three natural substrates 1-3were acetylated by both the wild type CALB and cp283. Product analysisof reactions with 3 by chiral gas chromatography (Agilent 6850 GCequipped with CycloSil-B chiral column) showed faster esterification ofsubstrate 3 by cp283 in comparison to wild type CALB. Equally important,the enantioselectivity was found uniformly high in both reactions.

Chiral tertiary alcohols and their esters (TAEs) are found in numerousnatural products and represent valuable building blocks for organicsynthetic chemistry. Their preparation by enzymatic kinetic resolutionas an alternative to inadequate synthetic methods has been contemplated,yet the majority of lipases that are successfully employed for theseparation of secondary alcohols show poor reactivity and at bestmediocre enantioselective for TAEs. The enzymes' inferior performance onTAEs is believed to result from steric limitation within the active siteof the catalyst. Structure comparison of lipases capable of hydrolyzingtertiary alcohols with non-active catalysts suggests a wideralcohol-binding pocket in the former which facilitates the binding ofthe larger tertiary group. The circular permutation methods of thepresent disclosure have identified new termini proximal to this bindingsite to test this hypothesis. Lipases capable of hydrolyzing tertiaryalcohols also carry a distinctive GGGX loop as part of the active site,believed to maximize the flexibility of the oxyanion-stabilizing pocket.In summary, no biocatalyst with satisfying enantioselectivity andactivity for even simple esters with tertiary alcohols have beenreported in the literature.

Rational engineering attempts and directed evolution have not yielded asuitable catalyst either. While the studies have highlighted some of theunderlying problems with the current lipases, little has beenaccomplished towards the exploration of new protein engineeringapproaches to redesign and extend lipase activity for tertiary alcohols.Although such conventional approaches have not produced mutants with thedesired activity and selectivity, alterations of the active site bindingpocket in permutated CALB of the present disclosure, or other permutedlipases and esterases according to the present disclosure, may be ableaccommodate these novel substrates.

Another potential application for these lipase variants is the synthesisof functional polymers. The high selectivity of CALB, together with itscatalytic activity in aqueous and organic media has made the lipase anappealing polymerization catalyst. For example, the enzyme is utilizedfor the ring-opening polymerization of cyclic lactones such as theseven-membered ε-caprolactone. Of interest is the enzyme's limitedcapability to hydrolyze smaller ring systems such as δ-valerolactone andγ-butyrolactone, presumably caused by the higher rigidity of the ringthat does not fit into the enzyme's active site. It is believed thatcircular permutation of the CALB can provide a catalyst with moreflexibility in accommodating monomeric substrates, expanding the rangeof polymer-building blocks that can be utilized.

As the reorganization of the active site binding pocket as a result ofthe circular permutation is a possible mechanism for the generation ofnovel hydrolase activity, circularly permuted CALB variants may alsohave novel catalytic activity. Catalytic promiscuity in lipases andother α/β hydrolases have been reported, and thus alterations in theactive site binding pocket as a result of circular permutation offer avery attractive mechanism for shifting enzyme substrate specificity.

Impact of circular permutation on protein structure and dynamics: Theconsequences of circular permutation on a protein's structural integrityand dynamics are not well understood, and little experimental work tothat end has been described in the literature. The above-described datademonstrate that permutation can have a beneficial effect on thecatalytic performance of CALB, yet the rational behind this observationis unclear. The data suggest that the new termini make significantcontributions to catalysis and that the observed rate enhancements maynot simply be attributed to faster product release alone. Thepreservation of enantioselectivity described above, as well as the minorchanges in K_(M) values in the variants shown in Tables 1 and 2 above,suggests that the substrate binding site remains largely intact despitethe cleavage of the backbone.

Thus, circular permutation is believed to have consequences on the localprotein environment. For example, cleavage of the peptide bond betweenamino acid 282 and 283 (cp283) may affect the local dynamics of the twosmaller but defined helical regions, or the permutation may result inthe “unraveling” of the helical regions, generating two disorderedtethers. To study the impact of circular permutation on proteinstructure on a molecular level, a series of biophysical experimentsbased on circular dichroism and fluorescence spectroscopy were designed.These studies are complemented by secondary protein engineering of theCALB variants.

A protein's secondary structure content can be estimated by far UV CDspectroscopy. The spectra analysis of selected CALB variants listed inTable 1 and Table 2 shows little structural changes in permutants withtermini in α7/9, as illustrated in the Far-UV circular dichrosim spectraof FIG. 7. In contrast, a clear decrease in the CALB variants' helicalcontent is observed when the protein termini are located in a 16/17 asshown in FIG. 8. The decreases in mean ellipticity at 195 and 222 nm areindicative of reduced helical content in the enzyme variants.Furthermore, a correlation between the declining helical content and theposition of the protein termini moving from cp289 to cp268 was noticed.These data suggest that elements of these helices which, uponpermutation, shift to the N-terminus may not fold into a definedsecondary structure. Such a trend of decreasing structural integrity isalso consistent with separate CD thermodenaturation experiments shown asthe inserts on FIGS. 7 and 8. Termini relocation from cp289 to cp268shows a steady decrease in T_(m) and a departure from a sharp two-statetransition to less-cooperative protein unfolding.

In searching for an explanation for the destabilization of theN-terminal helix fragment, it was noticed that connecting the wild typetermini with a six amino-acid linker forms of an extended loop near theamino terminus, as illustrated schematically in FIG. 9C. Large loops inproteins have been found to be thermodynamically unfavorable, decreasingthe thermostability of model proteins. While the increased loopflexibility showed little changes in folding behavior of the protein,its effect on the free energy of the protein may be accounted for by theentropic cost of loop closure. Assuming that this loop region does notadopt secondary structure that could stabilize the protein, this modelindicates the general trend in protein destabilization in connectionwith loop extensions. Thus, the CD results suggest that losses insecondary structure may accompany gains in catalytic activity of CALB.It is believed that these structural changes likely occur near thepermutation site. Thus, this extended loop provided an interestingtarget for secondary engineering, as discussed in greater detail below.

Among the functionally selected CALB variants, permutants with newtermini in α-helix 16 and 17 stand out in regard to the location of thebackbone cleavage and the observed catalytic rate enhancements. As thekinetic data for cp294 show that deletions in that region can bedetrimental to catalysis, the new termini likely play an important roletowards enzyme function, yet it remains unclear whether the fragments ofthe cleaved helices retain their secondary structures or becomeunstructured tethers.

Fluorescence spectroscopy can be used to clarify the impact of circularpermutation on the enzymes' structural integrity. Specifically,time-resolved fluorescence anisotropy experiments can be used toinvestigate the dynamics of the polypeptide chain at or near thecleavage site. Similar experiments on acetylcholinesterase havedemonstrated that site-specific fluorophor labeling in the protein canbe used to investigate the conformational changes upon substratebinding, as well as to probe the nano to microsecond dynamics ofselected regions in the apo-protein. With these methods, the flexibilityof fluorophors, attached to the side chain of a cysteine at or nearpermutation sites, can be measured. Serving as the “rigid” reference,the intact helix in wild type CALB provides little flexibility forlabels, resulting in slow signal decays. In contrast, the labeled C293residue in CALB(Δ301) (see FIG. 9) sits on a seven amino acid-longtether, minimizing conformational constraints and making it a referencefor a highly flexible protein chain.

In order to attach fluorophor labels in α17, multiple surface-exposedpositions were selected throughout the helix. For the second generationof mutants, three single cysteine mutants in positions A279, V286 andG288 have been created. These residues are located one or two helixturns away from the protein termini. As discussed below, the expressionof properly folded and active enzymes with a free cysteine have beenaddressed in activity assays with the truncated enzyme CALB(Δ301), whichhas an unpaired cysteine, yet can be overexpressed in Pichia pastoris atwild type levels.

Secondary engineering of CALB variants: Based on the interpretation ofthe CD data, discussed above and shown in FIGS. 7 and 8, the possibleconnection between declining protein stability and the approximately 40amino acid-long extended loop was tested by incremental truncation ofwild type CALB (FIG. 9B) and cp283 (FIG. 9D).

To explore the functional necessity of the extended unstructured regionin wild type CALB, a library of CALBs with incrementally truncatedC-terminus (FIG. 9B) was created. A comprehensive library of C-terminaltruncated CALBs was generated using ITCHY technology (Lutz S, OstermeierM, Benkovic SJ: Rapid generation of incremental truncation libraries forprotein engineering using alpha-phosphothioate nucleotides. NucleicAcids Res 2001, 29:E16, incorporated herein by reference in itsentirety) and underwent functional screening on tributyrin plates.Lipase genes from halo-forming colonies were analyzed by DNA sequencing.The results from this study show that the sixteen C-terminal amino acidsof CALB can be removed without loss of lipase function. The shortestCALB variant, CALB(Δ301), is currently undergoing detailed kinetic andbiophysical characterization. The truncation variants are named withrespect to the location of the new C-terminus in the truncated peptidesequence; thus, CALB(Δ301) is a native CALB having its new C-terminus atamino acid 301 (where 16 amino acids from the C-terminal end have beenremoved).

Separately, CALB(Δ301) can serve as a reference for fluorescenceanisotropy experiments described briefly above. As the truncation of theC-terminus removes C311, one of the natural six cysteines that formthree disulfide bonds in the wild type enzyme, CALB(Δ301) is left withan unpaired thiol in position 293, making it unique labeling site in theflexible C-terminus. Protein overexpression data show no interference ofthe free C293 with the folding of the active truncated enzyme.

Separately, an incremental truncation experiment was performed toidentify shorter versions of the newly created extended loop in cp283,which is shown schematically in FIG. 9D. Partial sequences, indicatingthe deleted amino acids, of several truncated variants of cp283 are alsoshown in FIG. 9. The cp/deletion variants are named with respect to thenumber of deleted amino acids. For instance, cp283 Δ11 indicates thatthe sequence is a variant of cp283 having 11 amino acids removed fromthe extended loop. The suffixes a, b, c, and so on, indicate differentvariants with the same number of deletions. Using CALB variant cp283 astemplate, a random library of ˜3×10⁶ lipase variants was created usingthe ITCHY technology as described in Example 11. Functional screening of˜40,000 colonies identified numerous colonies with lipase activity, andDNA analysis has identified several active proteins with deletions of upto eleven amino acids in the loop. Subsequent overexpression and kineticanalysis of some of these protein variants has confirmed higher thanwild type activity. For instance cp283Δ7a (as featured, in part, in SEQID NO: 20) was found to have increased activity over that of the nativeCALB.

In addition, analysis of these truncation variants of cp283 revealedthat truncation of the loop affected dimerization of the CALB variant,which was related to stability, as described in Example 11 andillustrated in FIGS. 10 and 11. Some of the truncation variants wereanalyzed for secondary structure content and thermostability, asillustrated in FIG. 11. Variants cp283-Δ2 to Δ7 showed increasing T_(M)and wild type-like secondary structure content, while variants cp283-Δ8to Δ11 showed decreasing T_(M) and loss of secondary structure content.Thus, it appears that truncations in the extended loop of CALB-283results in more native-like secondary structure and increased stability,due at least in part to an increased ability to form dimers.

The methods described herein using the concept of circular permutationand optionally followed by secondary engineering may be applied to otherlipases, esterases, hydrolases, and the other proteins and peptides andare not intended to be limited to the embodiment described herein or inthe examples below.

EXAMPLES

The following detailed examples are given to illustrate some preferredembodiments of the present disclosure and are not intended to limit itin any manner.

Example 1 Materials

Chemicals: Fluorogenic substrate 6,8-difluoro-4-methylumbelliferyloctanoate (DiFMU octanoate) and the reference standard6,8-difluoro-7-hydroxy-4-methylcoumarin (DiFMU) were purchased fromMolecular Probes (Eugene, Oreg.). p-Nitrophenyl butyrate (p-NB) waspurchased from Sigma (St. Louis, Mo.). Enzymes were purchased from NewEngland Biolabs (Beverly, Mass.) unless noted otherwise.

Strains and media: Pichia pastoris GS115 (his4) (Invitrogen, Carlsbad,Calif.) was used for the lipase expression. E. coli strainDH5α-E(Invitrogen, Carlsbad, Calif.) was used for all vectorconstructions. P. pastoris was grown in YPG medium (10 g yeast extract,20 g bacto peptone, 20 g glucose per liter). BMGY medium (10 g yeastextract, 20 g peptone, 13.4 g yeast nitrogen base, 0.4 mg biotin, 10 mlglycerol, and 100 ml 1 M potassium phosphate buffer, pH 6.0 per liter)and BMMY medium (10 g yeast extract, 20 g peptone, 13.4 g yeast nitrogenbase, 0.4 mg biotin, 5 ml methanol, and 100 ml 1 M potassium phosphatebuffer, pH 6.0 per liter) were used for protein expression. MD His⁻plates were used for selection of transformants (13.4 g yeast nitrogenbase, 0.4 mg biotin, 20 g dextrose, 15 g agar per liter). MM tributyrinplates were used to screen the lipase-secreting transformants (13.4 gyeast nitrogen base, 0.4 mg biotin, 5 ml methanol, 10 ml tributyrin, 15g agar per liter).

Example 2 Construction of wt-CALB Expression Vectors

The wild type calB (wt-calB) gene SEQ ID NO: 1 (having protein sequenceSEQ ID NO: 2) was isolated from Candida antarctica (ATCC strain #32657)by a two-step PCR amplification using the primers ZQ_CALBfor1(^(5′)-GAGGCTGAAGCTCATCATCATCATCATCATAGCAGCGGCCTTGTTCCA CGTCTACCTTCCGGTTCGGACCCT-^(3′)) (SEQ. ID NO: 5), ZQ_CALBfor2(^(5′)-CGCCTCGAGAAAAGAGAGGCTGAAGCT CATCATCATCATCATCAT-^(3′)) (SEQ. IDNO: 6), and ZQ_CALBrev (^(5′)-CGCGCGGCCGCTTAGGGGGTGACGAT GCCGGAGCA-^(3′)) (SEQ. ID NO: 7). The amplified gene included a (His)₆ tagfollowed by a thrombin cleavage site at the N-terminus of the lipasegene. Restriction enzyme recognition sites XhoI and NotI were alsointroduced into the 5′ and the 3′ ends respectively (recognitionsequence underlined). The PCR product was digested with XhoI and NotIand ligated to the vector pPIC9 (Invitrogen, Carlsbad, Calif.) digestedwith the same restriction enzymes. This construct (pPIC9-calB) broughtthe CALB gene under the control of the methanol inducible alcoholoxidase promoter (AOX1) and in frame with the α-factor secretion signalpeptide of Saccharomyces cerevisiae.

Example 3 Random Circular Permutation of calB

The wt-calB (SEQ ID NO: 1) was amplified by PCR using primersZQ_cpCALBfor (^(5′)-GGTACTAGTGGTGGCCTACCTTCCGGTTCGGACCCT-^(3′)) (SEQ. IDNO: 8) and ZQ_cpCALBrev (^(5′)-CGCACTAGTACCGCCGGGGGTGACGATGCCGGAGCA-^(3′)) (SEQ. ID NO: 9) harboring a SpeI site at both ends(underlined). After digestion with SpeI, 5 μg PCR fragment wascircularized at a concentration of 2.5 ng/μl with 90 Weiss units T4 DNAligase (Promega, Madison, Wis.) overnight at 16° C. This constructgenerated a circular calB with an 18-bp linker sequence (SEQ. ID NO: 4)that encodes Gly-Gly-Thr-Ser-Gly-Gly (SEQ. ID NO: 3) joining the naturalN- and C-terminals. The linker designed consisted of a six-amino acidpeptide, rich in glycine for flexibility and serine/threonine forhydrophilicity. After ethanol precipitation, the DNA was subjected toexonuclease III (0.4 units/μg DNA, Promega, Madison, Wis.) digestion at37° C. for 30 min to remove remaining linear DNA. The exonuclease IIIwas inactivated by heating at 65° C. for 15 min. The DNA was purified byQIAquick columns and eluted with 50 μl EB buffer.

Random relinearization of the circularized gene was performed by limiteddigestion with DNaseI (Roche, Indianapolis, Ind.) (RNaseI-free; 0.5milliunits/μg DNA) in 50 mM Tris.HCl, pH7.5, 1 mM MnCl₂, DNA (5 μg/ml)at room temperature for 15 min. The reaction was stopped by adding 10 μl0.5 M EDTA, and desalted by QIAquick columns (Qiagen, Valencia, Calif.)into elution buffer (10 mM Tris-HCl, pH 8.5). The linearized DNA wasrepaired using T4 DNA polymerase (Promega, Madison, Wis.) (1 unit/μgDNA) and T4 ligase (2 Weiss units/μg DNA) at room temperature for 1 h inT4 ligase buffer with the addition of dNTPs to a final concentration of150 μM. The linearized and cured DNA was recovered by agarose gelelectrophoresis.

Example 4 Creation of the pPIC9-cp-calB Library

The direct blunt-end ligation of cp-calB into the expression vectorpPIC9 was difficult due to the vector's size. Successful libraryintegration was instead achieved by using pAMB-CAT (Ambion, Austin,Tex.) as a shuttle vector. In preparation for library cloning, pAMB-CATwas modified to carry the N-terminal extensions (His tag, Thrombincleavage site, start codon) upstream from the calB cloning site plus astop codon immediately following the site of insertion. Therefore,PCR-amplified wild type calB (primers: ZQ_CALBfor1, ZQ_CALBfor2,ZQ_CALBrev) (SEQ. ID NOs: 5, 6, and 7, respectively) was digested withNotI/XhoI and ligated to the vector pAMB-CAT digested with the samerestriction enzymes. The resulting vector was amplified using primersZQ_pAMBfor (^(5′)-CCG

AGGCCTT GGAACAAGGCCGCTGCTATG-^(3′)) (SEQ. ID NO: 10) and ZQ_pAMBrev(^(5′)-CCG

TTATAAGCGGCCGCAAGCTTGTCG-^(3′)) (SEQ. ID NO: 11), which harbored a StuIand a PsiI site (underlined) as well as EcoRV sites (dashed lines)flanking both ends. The amplified vector was digested with EcoRV andligated with a segment generated from EcoRV digestion of pET-16b vector(Novagen, Madison, Wis.) to increase the size of the insert. Thisenabled subsequent digests to be monitored. Finally, the vector wasdigested with StuI and PsiI and the cp-calB library was incorporatedinto the vector by blunt-end ligation. Transformation of the plasmidinto electro-competent E. coli DH5α-E cells generated the pAMB-cp-ca/Blibrary (˜5×10⁵ members). The colonies were harvested and the plasmidwas isolated by QIAprep Spin Miniprep kit.

In a second cloning step, the cp-calB library was integrated in pPIC9.Purified pAMB-cp-ca/B was digested with NotI/XhoI and the segmentcontaining the cp-calB library was ligated to the pPIC9 vector digestedwith the same enzymes. Approximately 1.5×10⁶ colonies were obtainedafter transformation into electro-competent E. coli DH5α-E cells. Thetransformants were harvested and the plasmid was isolated using theQIAprep Spin Miniprep kit.

The two-step protocol yielded libraries consisting of 500,000 members.Given the theoretical library size of 317+6 (protein length plus peptidelinker), such library size virtually guaranteed that each member of thelibrary was represented at least once. The absence of any detectablebiases in the distribution of newly created protein termini wasconfirmed by DNA sequencing of 89 CALB genes of randomly picked librarymembers (FIG. 3). In addition to the expected permuted full-length CALBgenes, several library members that carried insertions and deletions ofone or more residues of the wild type protein were identified. In somecases, the manipulation of the gene sequences by PCR also introducedadditional sequence variation as a result of one or more nucleotidesubstitutions that can result in mutations of the original proteinsequence.

Finally, an important aspect for the analysis of the CALB variants wasthe transformation of the single cp-CALB gene library member per hostcell. In contrast to bacterial expression systems, Pichia pastorisincorporates the transformed plasmid and its content into itschromosomal DNA. While multiple integrations of a target gene per cellare advantageous for homogenous DNA samples, leading to higherexpression levels, the same is not the case for libraries. Theintegration of multiple library members in a single host's DNA coulddilute the functional properties of individual members. Further, hostcells, which show lipase activity, would require extensive secondarycharacterization to isolate the functional variant(s) in the sequencepool. Various transformation protocols, known to those of skill in theart, can be used. In the present experiments, electroporation producedthe highest fraction of single gene incorporations. Sequence analysisindicated that approximately 75-80% of the colonies carries only asingle library member.

Example 5 Library Screening

After digestion with SacI and ethanol precipitation, the pPIC9-cp-calBlibrary was transformed into electro-competent P. pastoris strain GS115(as described in Wu, S. et al., Biotechniques 2004, 36, (1), 152-4,which is hereby incorporated by reference) and plated on MM-tributyrinplates. Upon expressing a lipase variant, the yeast exports thepro-protein into the cell's surrounding media as defined by the attachedα-signal sequence. Following cleavage of the lipase's pro-sequence by anextracellular protease, functional library members will hydrolyze theemulsified tributyrin, a short-chain triglycerate, into water solubleproducts which creates a “clearing zone,” as illustrated in FIG. 4,around that particular colony. Tributyrin is considered an easysubstrate that can be utilized by the vast majority of known lipases.

Colonies appeared after four days of incubation at 30° C. Activecp-CALBs were identified by the formation of a clear halo surroundingthe respective host colony, as described in Gupta, R., et al.,Biotechnol Appl Biochem 2003, 37, which is hereby incorporated byreference. The growth of the cp-library on these screening platesproduced several hundred halo-forming colonies. These colonies werepicked and replated on MD and MM-trybutyrin plates to verify the lipaseactivity in a secondary screening, also illustrated in FIG. 4. Afterconfirmation of the lipase activity, the sequences of the correspondingcp-CALB genes were obtained by colony PCR and DNA sequencing usingprimers ZQ-pPIC9-for (^(5′)-TACTATTGCCAG CATTGCTGC-^(3′)) (SEQ. ID NO:12) and ZQ-pPIC9-rev (^(5′)-GCAAAT GGCATT CTGACATCC-^(3′)) (SEQ. ID NO:13).

Sequence analysis of 280 colonies led to the identification of 63 uniquecircular permutated CALBs, and the distribution of the selected variantswas visualized in FIG. 5 (outer circle lines). As the growth temperatureof the culture could potentially bias the outcome of the screeningexperiment by favoring proteins with higher thermostability, theseexperiments were conducted at room temperature, as well as 30° C. Nodifferences in the distribution were however detected. Furthermore, theimpact of the N-terminal His-tag on lipase variant screening andfunction was investigated. Protein libraries without the His-tag showedthe same distribution of functional permutants as the tagged variants.

The analysis of the current 63 functional lipase variants, and mappingof the new termini location on the tertiary structure of CALB (FIG. 6)gives raise to some interesting results. While a good number ofpermutations are located in surface loop regions as expected, FIG. 6indicates that the C-terminal region appears to be more susceptible tothe introduction of backbone cleavage without loss in function. Inparticular, the bent helix 16-17 of the cap domain can be cleaved atalmost every single amino acid. This result is even more exciting as thehinge region between the two helices covers a significant portion ofCALB's active site. The introduction of a backbone cleavage is thereforelikely to affect the catalytic performance of the enzyme. A secondregion with multiple permutations is the region of helix 7-9 thatconstitutes the lid region of CALB. Although smaller and less importantfor the function of CALB, this region is important for most lipases asit undergoes an important conformational change that activates thelipase. Surprisingly, permutations were also found in helix 2 that formspart of the oxyanion-binding pocket in the active site. All indicatedpermutation sites were confirmed by isolation of the corresponding gene,retransformation, verification of the halo formation, and repeated DNAsequencing.

Example 6 Protein Expression and Purification

The overexpression and purification of wild type CALB was performed asdescribed in Rotticci-Mulder, J. C. et al., Protein Expr Purif 2001, 21,(3), 386-92, which is hereby incorporated by reference. The sameprotocol was adopted for the isolation of circular permutation variantsof CALB. Briefly, pPIC9-ca/B was linearized by SacI digestion andelectroporated into P. pastoris cells (GS115). Aliquots were plated onMD His⁻ plates and incubated at 30° C. Colonies appeared on plates after2 days of incubation. A single colony was picked to inoculate 25 ml BMGYmedium and the culture was incubated at 30° C. until it reached an OD₆₀₀of 2-6. The cells were harvested and resuspended in BMMY medium to anOD₆₀₀ of 1. Protein expression was induced by addition of methanol to afinal concentration of 0.5% (v/v) every 24 hours. After 4 days ofincubation, the culture medium containing the lipase was separated fromthe cells by centrifugation (1500 g, 4° C., 10 min).

The His-tagged CALB was isolated from the clear supernatant via affinitychromatography on Ni-NTA agarose (Qiagen, Valencia, Calif.) using 2.5 mlresin per 100 ml supernatant. The column was washed with two columnvolumes of buffer 1 (20 mM imidazole, 300 mM NaCl, 50 mM NaH₂PO₄, pH8.0) and enzyme was eluted in two column volumes of buffer 2 (250 mMimidazole, 300 mM NaCl, 50 mM NaH₂PO₄, pH 8.0). All fractions wereanalyzed by SDS-PAGE and product-containing aliquots were pooled.Purified CALB was exchanged into storage buffer (150 mM NaCl, 50 mMK-phosphate, pH 7.0) by ultrafiltration (Amicon Ultra-4 centrifugalfilter unit; Millipore, Bedford, Mass.), and stored at 4° C. The proteinconcentration was determined spectrophotometrically at 280 nm (ε=3.3×10⁴M⁻¹ cm⁻¹), as described in Rotticci, D. et al., Biochim Biophys Acta2000, 1483, (1), 132-40, which is hereby incorporated by reference.

Alternatively, hydrophobic interaction chromatography (HIC) incombination with size exclusion chromatography was employed to purifyCALB to homogeneity as described above. The two-step purificationenables the rapid isolation of lipase variants whose His tag is notaccessible (circular permutants with termini in the protein's interiorregion) or has been removed all together. Addressing concerns that theHis-tag may interfer with the enzyme function, a second selection ofexperiments we performed with same calB library without affinity tag.The DNA sequence analysis of functional candidates indicated that thelocation and distribution of permutation sites in functional CALBvariants was the same as shown in FIG. 5. For the HIC purificationroute, the clear culture supernatant was mixed with 2 M (NH₄)₂SO₄solution and 1 M K-phosphate buffer (pH 7.0) to a final concentration of1 M and 50 mM respectively. The protein samples were then loaded on aHIC column (7 ml butyl-sepharose 4 resin (AmershamBiosciences,Piscataway, N.J.), pre-equilibrated with 1 M (NH₄)₂SO₄, 50 mMK-phosphate buffer (pH 7.0) (buffer 4). The column was rinsed with 4volumes of buffer 4, followed by a stepwise reduction of (NH₄)₂SO₄ inthe phosphate buffer (0.2 M increments, 4 column volumes per step).Lipase activity in the eluant was monitored via p-NB hydrolysis (seebelow) and fractions containing the desired activity were pooled andconcentrated by ultrafiltration (Amicon Ultra-15 centrifugal filterunit; Millipore, Bedford, Mass.). According to SDS-PAGE, the elutedprotein has >85% purity. Further removal of contaminants was possible bygel filtration on a Superdex-200 10/300 GL column (AmershamBiosciences,Piscataway, N.J.), using 50 mM K-phosphate buffer (pH 7.0) containing150 mM NaCl. SDS-PAGE analysis of the final product showed >95% purity.

Example 7 Activity Assays

Lipase activity was determined by measuring the initial hydrolysis rateof p-NB and DiFMU octanoate at room temperature on a Synergy-HTmicrotiterplate reader (Bio-Tek Instruments, Winooski, Vt.). Stocksolutions of p-NB (200 mM) and DiFMU octanoate (3 mM) were prepared inDMSO. p-NB hydrolysis over a substrate range of 0-1.6 mM was measured in50 mM K-phosphate buffer (pH 7.5) at 400 nm (

for p-NB=13260 M¹ cm⁻¹) as described in Bender, M. L. et al., J Am ChemSoc 1968, 90, (1), 201-7, which is hereby incorporated by reference. Therate of DiFMU octanoate hydrolysis was determined by measuring the DiFMUformation over a substrate range of 0-12 μM in 50 mM K-phosphate buffer(pH 7.0) at an excitation/emission wavelength 360/460 nm. Kineticconstants were calculated by fitting the initial rates to theMichaelis-Menten equation using the Origin® software (version 7;OriginLab Corporation). The results are presented in Tables 1 and 2,above.

Example 8 Large-scale Lipase Overexpression for Biochemical &Biophysical Studies

A batch-fermentation protocol for overexpressing CALB in Pichia pastoriswas established and implemented. The experiments with wild type andpermutated CALBs consistently yield ˜600 mg protein per liter of culturemedium. The target protein is secreted into the culture medium and canbe isolated with >95% purity via one-step purification over a weakion-exchange resin.

For experiments in organic solvents, CALB is immobilized on Lewatit VPOC1600 (Sybron Chem. Inc) and the amount of active lipase on the resin isquantified via active site titration with the suicide inhibitors (asdescribed in Rotticci D., et al., An active-site titration method forlipases. Biochim Biophys Acta 2000, 1483:132-140; and Fujii R,Utsunomiya Y, Hiratake J, Sogabe A, Sakata K: Highly sensitiveactive-site titration of lipase in microscale culture media usingfluorescent organophosphorus ester. BBA-Molecular and Cell Biology ofLipids 2003, 1631:197-205. Such a suicide inhibitor,methoxy-4-methylumbelliferyl hexylphosphonate, has been synthesized andsuccessfully used to determine enzyme loads on the resin.

Example 9 Kinetic Analysis of Lipase Catalyzed TransesterificationReactions

Transesterification of 6-methyl-5-hepten-2-ol with vinyl acetate wasperformed in hexane at 23° C. Each 1 ml reaction mixture contains from1-10 mg immobilized enzyme, 50 mM internal standard6-methyl-5-hepten-2-one and varying amount of racemic6-methyl-5-hepten-2-ol (25˜500 mM). The mixture was incubated at 23° C.for 30 minutes and the reaction was initiated by the addition of 0.5mmol vinyl acetate. Samples at different time points were taken todetermine the initial reaction rates. For each reaction at least fivesamples were taken, and the overall conversions was limited to 5% of thesubstrate. The samples were analyzed by gas chromatography G6850(Agilent Technologies) installed with a Cyclosil-B column (length 30 m,i.d. 0.32 mm, film 0.25 mm, Agilent) connected to a flame ionizationdetector. Hydrogen was used as the carrier gas, and the temperatureprogram was: 70° C. for 1 min, 2° C./min to 90° C. and hold for 3 min,then 10° C./min to 120° C. and hold for 3 min. The retention time was12.2 min for S-6-methyl-5-hepten-2-ol and 12.8 min for its R-enantiomer.

Transesterification of 3-hydroxy tetrahydrofuran with vinyl acetate wasperformed the same except that acetonitrile was used as solvent. Thetemperature program for GC analysis was: 65° C. for 5 min, 2° C./min to90° C., then 10° C./min to 120° C.

Example 10 Incremental Truncation of C-terminus of Wild Type CALB

Wild type CALB gene (SEQ ID NO: 1) was PCR amplified using primersCALB_for_hisfree (5′-CGCCTCGAGAAAAGAGAGGCTGAAGCTCTACCTTCCGGTTCGGACCCTGCC-3′) (SEQ ID NO: 24) and ZQ_CALB_rev (5′-CGCGCGGCCGCTTAGGGGGTGACGATGCCGGAGCA-3′) (SEQ ID NO: 7). The PCR product wasdigested with NotI and XhoI and ligated into the vector pAMB-CATdigested with the same restriction enzymes. The plasmid was linearizedby EcoRI digestion, and the incremental truncation library was generatedfollowing the protocol of Marc Ostermeier and Stefan Lutz (Methods inmolecular biology, Vol 231, 129-142). In detail, the linearized plasmidwas amplified by Taq DNA polymerase using primers Trunc_for(5′-GAGCTCCGTCGACAAGCTTGCGG-3′) (SEQ ID NO: 28) and Trunc_rev(5′-GGATGAGCATTCATCAGGCGGGCA-3′) (SEQ ID NO: 29). The 50 μl PCR reactionmixture contained 175 μM dNTP/25 μM αS-dNTP (dNTP:αS-dNTP=7:1). Afterpurification with Qiagen's QIAquick PCR purification kit, the PCRproduct was digested by Exonuclease W (120 units/μg DNA) at 37° C. for30 min. The reaction was quenched by the addition of 5 volumes of PBbuffer and purified by QIAquick PCR purification kit. The 5′-overhangwas removed by incubation with mung bean nuclease (2.5 units/μg DNA, DNAconcentration 0.1 μg/μl) at 30° C. for 30 min, and the DNA was purifiedby Qiagen spin columns. Then the purified DNA was treated with Klenowpolymerase to repair the sticky ends (1 units/μg DNA, DNA concentration0.1 μg/μl, 25° C. for 15 min and 75° C. for 20 min). After purificationby Qiagen spin column, the DNA was digested with XhoI, and sizeselection (fraction between 750 bp and 1 kb) was performed afterwards bygel extraction.

The extracted DNA was ligated into a modified vector pAMB-pET digestedwith Psil and XhoI, and transformed into E. coli DH5α cells. Around1.5×10⁵ colonies were obtained. The cells were harvested and the plasmidwas purified by Qiagen miniprep kit. After digestion the plasmid withNotI and XhoI, the fraction containing CALB gene fragments was extractedand ligated into the plasmid pPIC9 digested with the same enzymes. Theligation mixture was again transformed into DH5α, and a library of 1.2million colonies was obtained. The plasmid was purified, digested withSacI, transformed into Pichia Pastoris strain GS115 and plated onMM-tributyrin plates. Active library members were visualized by halosaround the colonies. Those colonies were picked and submitted to DNAsequencing.

TABLE 3 Activity of truncation members CALB Truncation Peptide Activity(Y/N) Δ296 N Δ288 N Δ277 N Δ254 N Δ249 N Δ301 Y Δ302 Y Δ303 Y Δ304 YΔ305 Y Δ306 Y Δ307 Y Δ308 Y Δ309 Y Δ310 Y Δ312 Y Δ313 Y Δ314 Y

Example 11 Incremental Truncation of the External Loop of cp283

In the present example, incremental truncation of the extended loop ofCALB-cp283 was performed to evaluate the effect on the thermostabilityof the variants. The gene encoding the peptide sequence of cp283 (SEQ IDNO: 14) was put into the vector pAMB-CAT using NotI and XhoI restrictionsites. Then the plasmid containing cp283 gene was linearized by SpeIdigestion (which is within the six amino acid linker between natural C-and N-termini). The linearized plasmid was amplified by Taq DNApolymerase using primers ZQ_cpCALB_for (5′-GGTACTAGTGGTGGCCTACCTTCCGGTTCGGACCCT-3′) (SEQ. ID NO: 8), ZQ_cpCALBrev(5′-CGCACTAGTACCGCCGGGGGTGACGATGCGGG AGCA-3′) (SEQ ID NO: 9), and spikeddNTPs (dNTP:αS-dNTP=7:1). The incremental truncation library wasgenerated the same way as the C-terminal truncation library, except thatintramolecular ligation was performed after the Klenow polymerasetreatment (DNA concentration: 2.5 ng/μl, 16° C. overnight). The ligationmixture was concentrated by ethanol precipitation and electroporatedinto DH5α. About 3 million colonies were obtained. Purified plasmid wassubjected to NotI and XhoI digestion, and the DNA fragment between 750and 1000 bp was extracted from agarose gel. The fragment was ligatedinto the vector pPIC9, and the following procedure was the same as theC-terminal truncation library. Partial sequences, showing the locationof deleted segments, of some the members of the cp283 truncation libraryare illustrated in FIG. 9.

Functional variants were identified by screening for tributyrinhydrolysis. It was found that up to 11 amino acids (approximately 25% ofthe extended loop) could be deleted without the loss of function. Allvariants up to cp283Δ11 retain the p-nitrophenol butyrate hydrolysisactivity of CALB cp283 (data not shown). The linker region and wild typeC-terminal region were found to be more tolerant to truncation than thewild-type N-terminal region. As shown in FIG. 11, variants cp283Δ2 to Δ7demonstrated increasing T_(M) and wild type-like secondary structurecontent, while cp283Δ8 to Δ11 showed lower T_(M) and loss of secondarystructure content. This abrupt transition at Δ7/Δ8 may reflect criticalloop size for proper orientation of the cp283 N-terminus.

Gel filtration analysis was perfomed to assess the quaternary structureand aggregation of truncated cpCALB variants, to determine whetherover-truncation of the loop and removal of disulfide-forming Cys295could affect stability and lead to aggregation. As illustrated in FIG.10, this analysis revealed that loop truncation in the variants affectedthe dimerization ability of the peptide. The dimeric form of CALBs wasfound to be stable and can be isolated for additional characterization.Analysis of cp283Δ4 monomer and dimer forms demonstrated that bothprotein forms have high catalytic activity, but the enhancedthermostability appears to be due to dimerization. It is believed thattruncation of the extended loop allows proper adjustment of loop lengthto optimize dimerization by domain swapping of N-terminus in cp283Δ7.

The results of the present study suggest that amino acid deletions inthe extended loop of the CALB linker region results in more native-likesecondary structure and increased stability. Furthermore, it appearsthat the increase in stability is not necessarily due to lower entropyof loop closure, but may be attributed to the increased ability todimerize in variants with optimal loop size and configuration.

It should be emphasized that the above-described embodiments of thepresent disclosure, particularly, any “preferred” embodiments, aremerely possible examples of the implementations, merely set forth for aclear understanding of the principles of the disclosure. Many variationsand modifications may be made to the above-described embodiment(s) ofthe disclosure without departing substantially from the spirit andprinciples of the disclosure. All such modifications and variations areintended to be included herein within the scope of this disclosure, andthe present disclosure and protected by the following claims.

Example 12 Circular Permutation Effects on Enantioselectivity andStructure

Random circular permutation generates small variations in polypeptidechain length, resulting from the insertion and deletion of amino acidsat the new termini. While some of the example above show that neithermodification of the protein sequence seems to affect function per se,potential effects on stability and overall catalytic efficiency couldnot be ruled out. Therefore, in the present study, the gene sequences ofall previously characterized CALB variants were corrected, making themuniform in size with wild type protein by removing amino acidduplications and filling in missing residues with gene-specific primers.All variants used in this study were 323 amino acids in length, 317residues from the native enzyme plus 6 amino acids from the linkersequence. Furthermore, the N-terminal His-tag was removed to eliminatethe potential for interference with catalysis. The tag was initiallyused for protein purification, yet changes in the experimental protocolhave made its presence optional.

The present study included ten CALB variants. Six variants had newtermini in the cap region, the lid permutants cp144, cp148, and cp150,as well as the helix 16/17 permutants cp283, cp284, and cp289. This setof enzymes was complemented with three additional helix 16/17 variants(cp268, cp277, cp278). The combination of these ten enzymes represents aset of biocatalysts with permutation sites along the entire length ofthe two surface-exposed helices. Furthermore, cp193 was included in thisstudy, a variant with its termini near Asp187 of the catalytic triad, toassess the effects of backbone cleavage near residues of the catalytictriad.

Kinetic Characterization with Reference Substrates

Given the size adjustments to the previously characterized CALB variantsplus the addition of new enzymes for this study, all ten enzymes werecharacterized with reference substrates p-nitrophenol butyrate (p-NB)and 6,8-difluoro-4-methylumbelliferyl (DiFMU) octanoate (see Tables 1and 2, in the detailed description above). The kinetic properties ofcp144, cp148, and cp150 remained unchanged from previous examples.Similarly, cp283-289 show little deviation from previous data for p-NBhydrolysis. In contrast, the removal of the termini extensions in cp283and cp284 resulted in an additional ten-fold improvement in DiFMUoctanoate turnover (see cp283a and cp284a in Tables 1 and 2 in thedetailed description above), raising the catalytic efficiency of thesetwo variants over wild type CALB by 175 and 141-fold respectively. Inboth cases, the higher specificity can be attributed exclusively tohigher rate constants as the KM values are the same as wild type. It isbelieved that these gains in activity could be linked to reduced stericinterference in the active site binding pocket or reflect changes in theconformational flexibility of these regions. Interestingly, cp289 withits termini shifted by 5 amino acids to a position outside helix 17shows no significant change in catalytic performance, maintaining highactivity for DiFMU octanoate with or without termini extensions.

Enantioselectivity of Circular Permuted CALB

Significant rate enhancements in engineered enzymes are frequentlyreported, yet often come at the expense of a catalyst'senantioselectivity. Furthermore, the reactivity of the leaving groups inthese two reference substrates has raised questions as to the enzymes'performance with regular, unactivated substrates. To address bothconcerns, a second series of kinetic experiments were conducted,comparing the esterification and transesterification of five chiralalcohols and carboxylates with wild type CALB and cp283, the lipase withthe highest rate acceleration in the previous tests.

Milligram quantities of both enzymes could be obtained by scaling upprotein overexpression in Pichia pastoris and immobilizing the secretedlipase on weak ion-exchange resin as described in the experimentalsection. The immobilized enzyme was quantified by active site titrationassay using the suicide inhibitor methyl 4-methylumbelliferylhexylphosphonate. Upon reacting in the lipase active site, the inhibitorreleases a stoichiometric amount of highly fluorescent4methylumbelliferone (4-MU), which can be used to calculate the enzymeload on the resin. The immobilized enzymes were used to catalyze thedescribed esterification and transesterification reactions in organicsolvents.

Based on wild type CALB's substrate preference and highenantioselectivity for secondary alcohols, three racemic secondaryalcohols with substituents of various size were chosen as testsubstrates.

The kinetic constants for the transesterification reaction of3-hydroxy-tetrahydrofuran (1), 6-methyl-5-hepten-2-ol (2), and1-(−1-naphthyl)ethanol (3) with vinyl acetate were measured (FIG. 12,Table 2). The results indicate that circular permutation does not affectthe enantiomeric preference of CALB. Both wild type enzyme and cp283strongly favor the (R)-isomers of 2 and 3. The GC analysis of thereaction mixture containing the (S)enantiomer showed no detectableproduct formation over the length of the assay. The reported minimumenantiomeric ratio (E-values) for these two substrates were estimatedbased on the instrument's detection limit. In the case of 1, the twoenzymes switched to (S)-isomer preference but did not show superiorenantioselectivity. The result can be explained by the symmetry of thesubstrate's oxolane ring which makes it difficult for CALB and otherlipases, which rely on size differences of the two substituents on thealcohol moiety, to distinguish the two stereoisomers. For all threesubstrates, the calculated E-values for the permuted enzyme were similarto wild type CALB, confirming that the enantioselectivity of the lipasewas not compromised as a result of circular permutation. Equallyimportant, the kinetic data for cp283 showed little deviation from thewild type enzyme. For 1, a two-fold higher KM but similar turnover rateresulted in a slightly lower performance constant for cp283 than wildtype CALB. In contrast, the engineered variant and wild type enzyme hadvirtually identical Michaelis-Menten binding constants for 2 and 3.Similar to the results with the two reference substrates, cp283 showednoticeably higher turnover rates for 2 and 3, raising kcat/KM by up tothree-fold.

Separately, the CALB-catalyzed esterification of two chiralcarboxylates, 2-phenyl propionic acid (4) and2-(3-fluoro-4-phenyl-phenyl)propionic acid (flurbiprofen, 5), withn-propanol was studied (FIG. 12, Table 2). Consistent with the resultsfor the chiral secondary alcohols, both enzymes favor the(R)-enantiomers of 4 and 5. Although both stereoisomers are turned over,a combination of elevated KM and lower kcat values results in thediscrimination against the unfavorable (S)-isomer in both enzymes.Furthermore, changes in the individual performance constants lead tooverall two-fold improved E-values for cp283. Finally, the comparison ofthe kcat/KM values for the two enzymes with the respective substratesshowed a 2 to 4-fold higher performance constant for the engineeredenzyme over wild type CALB. While the catalytic activity andenantioselectivity of cp283 is improved, the underlying cause for theperformance enhancement might differ for the two substrates. Thecatalytic gains for 4 seem to originate largely from a lower KM valuefor the (R)-isomer, while the higher kcat/KM for 5 results from anincrease in catalytic activity for the preferred enantiomer.

In summary, the results from these kinetic experiments demonstrate thatcircular permutation of CALB does not compromise the enantioselectivityof the biocatalyst. To the contrary, improvements in enantioselectivityplus elevated catalytic efficiency were observed for a number of testedsubstrates. As such, the experiments confirm the above results thatcircular permutation in selected portions of CALB can raise the enzyme'scatalytic activity for a substrate. Cp283's consistently higher turnoverrate and improved enantioselectivity, even for unactivated substrates,demonstrates the potential of CP in improving an existing biocatalyst.

Structural Consequences of Circular Permutation

In parallel with the kinetic analysis of the above CALB variants, theimpact of circular permutation on protein structure were also explored.Cp193 was excluded from these studies due to protein instability. Ininitial experiments, the effects of termini relocation on the tertiarystructure of CALB were assessed by intrinsic tryptophan fluorescence,using its seven native indole side chains for detecting changes intertiary structure. In addition, this study looked for changes inprotein folding and packing with 1-anilino-8naphthalene sulfonate (ANS),an environmentally sensitive hydrophobic fluorescence dye. Both analysesshowed no significant spectral deviation from wild type CALB, consistentwith overall compact, native-like tertiary structures for all thevariants (data not shown). These findings reaffirm that CP generally haslittle impacts on the overall protein tertiary structure. Insteadstructural changes usually are concentrated in two regions, the nativeand the newly created termini.

Circular dichroism (CD) spectroscopy in the far UV range revealedsignificant changes in the secondary structure content of circularpermuted CALB variants (FIGS. 7 and 8, discussed above). All permutedproteins maintained a mixed α-helix/β-sheet spectral signature, yettheir mean residue ellipticity (Θ) at 193 nm was consistently lower andthe minima at 209 nm and 222 nm lost intensity compared to wild typeenzyme. These observations are consistent with a decline in helicalcontent. The data interpretation was confirmed in secondary structuremodels of the MRE curves by DICHROWEB (FIG. 13, Table 3). Accompanyingthe observed decline in helical content, thermo-denaturation studies ofthese variants in the CD spectropolarimeter also showed a significantdrop in the temperature of unfolding from 53° C. for wild type CALB to,in the worst case, 34° C. for cp277 (FIG. 13, Table 3). Permutations inthe lid region (cp144-cp150) appear less destabilizing, causingdifference in TM values of only 3 to 7° C., while TM values for CALBvariants with new termini in helix 16/17 (cp268-cp289) fell by 12 to 19°C., depending on the termini location.

More interestingly, the TM values show a correlation with the far UV CDresults and kinetic data for the reference substrates. This data setindicates that placing α16/17 at the protein's C-terminus, as is thecase for cp289, seems less detrimental to structure and function thanpositioning the helix at its N-terminus as in cp268. It is believed thatin cp289, the glycine-rich linker, connecting the native termini,creates an extended, structurally poorly-defined region at the newN-terminus whose proper organization in the enzyme tertiary structure isnot critical for catalytic function. Such an interpretation is supportedby incremental truncation experiments, discussed above, showing thatwild type CALB tolerates deletion of up to 16 amino acids at its nativeC-terminus without loss of function. Helix 16/17 in cp289 forms the newC-terminus and, as suggested by the enzyme's native-like CD signature,can assume a defined secondary structure. In contrast, cp268 representsthe other extreme where helix 16/17 is positioned at the N-terminus,connected to the rest of the protein via the extended linker. It isbelieved that the higher flexibility of this linker region complicatesthe proper orientation of the helix in this CALB variant, negativelyaffecting not only overall protein stability and helix formation, butconsequently also catalytic function.

In separate experiments, the idea of substrate-induced conformationalchanges was tested. Speculating that the presence of a substrate in theactive site could stabilize the termini regions and lead to greaterstructural organization, the spectroscopic properties of wild type CALBand cp283 were measured after derivatization with the suicide inhibitormethyl 4-methylumbelliferyl hexylphosphonate. The presence of the boundinhibitor substantially raised the TM for both enzymes to 68 and 63° C.respectively, yet left the far-UV CD signatures unchanged (data notshown). These results suggest the absence of direct interactions betweenthe substrate and the new termini region. Not unexpected, enzymeimmobilization also increases the stability of these CALB variants. Theesterification experiments of 4 and 5 were carried out at 50° C. withoutnoticeable reduction in enzyme activity after more than an hour atelevated temperature.

Discussion

Circular permutation of CALB can generate engineered enzymes with over100-fold enhanced performance constants without compromising thebiocatalyst's enantioselectivity. These observations demonstrate that CPdoes not dismantle the active site but seems to introduce more subtleconformational changes that benefit catalysis when the termini arelocated in proximity to the active site. In contrast, backbone cleavagethat affects the proper geometry of the catalytic triad will bedetrimental to function as seen for cp193 and cp44/cp47. Furthermore,the fluorescence and CD spectroscopy data support the originalhypothesis that the termini relocation impacts mostly local proteinstructure near the old and new termini.

Focusing on the old and new termini regions in the most active CALBvariants with termini in the helix 16/17 region, a correlation betweencatalytic activity, stability and position of the backbone cleavage sitewas noted. Fragments of helix 16/17 on the N-terminal portion of theCALB variants appear to lack stability due to the adjacent extended loopstructure consisting of the native termini and the flexible six-aminoacid linker. Such extended loops in proteins have been shown to bedestabilizing to the protein overall and could explain the reduction onthe TM values of these variants, as explored in more detail in Example11, above.

Materials and Methods

Chemicals: Fluorogenic substrate 6,8-difluoro-4-methylumbelliferyloctanoate (DiFMU octanoate) and the reference standard6,8-difluoro-7-hydroxy-4-methylcoumarin (DiFMU) were purchased fromMolecular Probes (Eugene, Oreg.). p-Nitrophenyl butyrate (p-NB),6-methyl-5-hepten-2-ol, 3hydroxytetrahydrofuran, 1-(1-naphthyl)ethanol,flurbiprofen, and phenylpropionic acid were purchased from Sigma (St.Louis, Mo.). Enzymes were purchased from New England Biolabs (Beverly,Mass.) unless noted otherwise.

Strains and media: E. coli strain DH5α-E (Invitrogen, Carlsbad, Calif.)was used for all vector construction. Bacteria were grown under standardconditions in Luria-Bertani (LB) liquid media or on LB agar platessupplemented with the appropriate antibiotics. Lipases wereoverexpressed in Pichia pastoris GS115 (his4) (Invitrogen, Carlsbad,Calif.). P. pastoris was cultured in YPG medium (10 g yeast extract, 20g bacto peptone, 20 g glucose per liter). For protein overexpression, weused BMGY medium (10 g yeast extract, 20 g peptone, 13.4 g yeastnitrogen base, 0.4 mg biotin, 10 ml glycerol, and 100 ml 1 M potassiumphosphate buffer, pH 6.0 per liter) and BMMY medium (10 g yeast extract,20 g peptone, 13.4 g yeast nitrogen base, 0.4 mg biotin, 5 ml methanol,and 100 ml 1 M potassium phosphate buffer, pH 6.0 per liter).

Construction of vectors: To express CALB variants with defined termini,the genes of selected lipases in the random circular permutation librarywere PCR amplified with the corresponding primers. The PCR products weredigested with XhoI and NotI and ligated to the vector pPIC9 (Invitrogen,Carlsbad, Calif.) digested with the same restriction enzymes. Followingtransformation into E. coli DH5α-E, plasmids were isolated and thecorrect calb sequences confirmed by DNA sequencing. Protein expression,purification & standard activity assays: The laboratory-scaleoverexpression and purification of wild type CALB and the variants wasperformed as previously described in the Examples above.Spectrophotometric assays to measure lipase activity with the referencesubstrates p-NB and DiFMU octanoate were performed as described. Allexperiments were performed in triplicate. Kinetic constants werecalculated by fitting the initial rates to the Michaelis-Menten equationusing Origin7 (OriginLab, Northhampton, Mass.).

CD analysis: Far-UV circular dichroism (CD) spectra were obtained usinga J-810 spectropolarimeter (Jasco, Easton, Md.). Spectra were recordedat 10° C. from 260-190 nm (0.5 nm increments) using a 0.1 mm pathlengthcell, 20 nm/min scan rate, 4 s response time, and 2 nm bandwidth.Proteins were analyzed at concentrations of 1 mg/ml (as determined by UVabsorbance at 280 nm; ε=33000 M⁻¹ cm⁻¹) in potassium phosphate buffer(50 mM, pH 7). Recorded data represent the means of five scans. Spectrawere corrected for buffer absorbance and converted to mean residueellipticity ([θ]mrw). Thermal denaturation was monitored by followingthe ellipticity at 222 nm at a 1° C./min heating rate from 10 to 80° C.

P. pastoris fermentation: Fermentation was performed in a 5-liter NewBrunswick BioFlow 3000 fermenter (New Brunswick Scientific, Edison,N.J.). The reaction conditions and media composition were chosen basedon Invitrogen's standard protocol for Pichia pastoris fermentation.Briefly, the glass fermentation vessel containing half the workingvolume of fermentation basal salts medium was sterilized. Followingsterilization, the pH of the medium was adjusted to 5.0 and PTM1 tracesalts were added. Fermentation conditions for Mut+GS115 transformantswere set as following: 30° C., 500 rpm agitation, DO 30%, pH 5. Thereactor was inoculated with 10% of the initial fermentation volume. Uponreaching a cell wet-weight of 180 g/L, the culture was induced withmethanol for 2 days. Cells were harvested and the supernatant wascollected and filtered using Millipore 0.22 μm filter.

Protein Immobilization: Ion exchange resin Lewatit VP OC 1600 (Lanxess,Pittsburgh, Pa.) was used for lipase immobilization. Resin waspre-washed with ethanol and dried under vacuum. For immobilization, 1 gresin was repeatedly incubated with 50 ml aliquots of filteredfermentation supernatant in a head-over-head shaker at 4° C. Uponreaching saturation of the resin as detected by increasing enzymeactivity in the supernatant, the resin was washed with 50 mM potassiumphosphate buffer (50 mM, pH 6.0), dried under vacuum and stored at 4° C.

Synthesis of Methyl 4-methylumbelliferyl hexylphosphonate: The synthesiswas performed as described in R. Fujii, Y. Utsunomiya, J. Hiratake, A.Sogabe, K. Sakata, Biochim. Biophys. Acta Mol. Cell. Biol. Lipids 2003,1631, 197-205; D. Rotticci, T. Norin, K. Hult, M. Martinelle, Biochim.Biophys. Acta 2000, 1483, 132-140 (which is hereby incorporated byreference herein) with the exception of the use of the fluorophor 4-MUas leaving group instead of the chromophore 4-nitrophenol, improving theinhibitor's stability and lowering the detection limit of the activesite titration.

Experimental Procedure: Tetrazole (43.8 μmol, 0.45 M in acetonitrile, 97μl), methanol (4.38 mmol, 177 μl), and diisopropylethylamine (4.93 mmol,858 μl) were added to 30 ml toluene under argon. The solution was cooledto 6° C. before adding hexylphosphonic dichloride (4.93 mmol, 844 μl)dropwise. The solution was then warmed to room temperature and stirredfor 4 h. 4-MU (4.38 mmol, 0.7712 g) was dissolved in a mixture oftoluene (3 ml) and diisopropylethylamine (4.93 mmol, 858 μl) and addedto the solution. After stirring the reaction overnight at ambienttemperature, the solid was filtered off and washed with toluene and thecombined filtrate evaporated. A silica gel column eluted with methylenechloride:ethyl acetate (3:2) and preparative TLC plates withhexane:ethyl acetate (1:1) were used to purify the product, yielding 165mg (11% yield) of a slightly cloudy liquid. ¹H NMR (CDCl₃). δ, ppm: 0.89(t, 3H), 1.30 (m, 4H), 1.42 (m, 2H), 1.69 (m, 2H), 1.94 (m, 2H), 2.43(s, 3H), 3.83 (d, 3H), 6.25 (s, 1H), 7.18 (s, 1H), 7.23 (d, 1H), 7.57(d, 1H). ¹³C NMR (CDCl₃). δ, ppm: 14.1, 18.9, 22.4, 24.9, 26.3, 30.3,31.3, 53.2, 109.1, 114.2, 117.1, 117.2, 126.0, 152.1, 153.4, 154.6,160.7. ³¹P NMR (CDCl3). δ, ppm: 29.5.

Active site titration: The amount of active lipase immobilized on resinwas determined by active site titration assay. Briefly, stock solutionof Methyl 4-methylumbelliferyl hexylphosphonate (30 μl, 0.3 mM inacetonitrile) was diluted with acetonitrile (970 μl) and enzyme-resin(1-15 mg) was added. The reaction was incubated on a head-over-headshaker at ambient temperature for 7 days until the daily fluorescencereading (ex: 360/em: 460 nm) indicated that the reaction had reachedequilibrium. The released fluorophor was quantifiedspectrophotometrically and the amount of active lipase calculated basedon the linear relationship between immobilized enzyme and 4-MU insolution.

Kinetic analysis & enantioselectivity of immobilized lipases: The rateof transesterification of chiral alcohols 1-3 with vinylacetate wasperformed in 2-ml reaction volume containing 1-10 mg of enzyme resin.Product formation was monitored by chiral GC analysis. Specifically,reactions with 2 were performed in cyclohexane at room temperature,using 50 mM internal standard (6-methyl-5-hepten-2-one) and between 25mM to 1.2 M of substrate. After 30 min preincubation, the reaction wasinitiated by adding vinyl acetate (200 μl, [final]=1.2 M). At least fivesamples over a period of 1 to 6 minutes were taken, limiting overallsubstrate conversions to <5%. The samples were analyzed by GC(G6850-system; Agilent Technologies, Santa Clara, Calif.), using aCyclosil-B column (length 30 m, i.d. 0.32 mm, film 0.25 mm) connected toa FID. Hydrogen was used as the carrier gas, and the temperature was 75°C. for 30 min. The retention time for S-2 and R-2 was 17.1 min and 15.8min respectively. Transesterification of R/S-1 with vinyl acetate wasperformed in acetonitrile. The concentration of the internal standardwas lowered to 10 mM. The temperature program for GC analysis was: 65°C. for 5 min, 2° C./min to 90° C., then 10° C./min to 120° C. Theretention time for R-1 and S-1 was 15.1 min and 16.9 min respectively.The reaction of R/S-3 with vinyl acetate was also performed inacetonitrile with 10 mM benzophenone as internal standard. The GCtemperature program was 160° C. for 40 min. The retention time for S-3and R3 was 16.5 min and 18.3 min respectively.

The esterification reaction of chiral carboxylates 4 and 5 with1-propanol was performed in 4-methyl-2-pentanone at 50° C. Each 2-mlreaction mixture contained 10-40 mg enzyme resin, 10 mM 4′-methoxyacetophenone (for 4) or 5 mM benzophenone (for 5) as internal standard,as well as 50-600 mM of substrate. The mixture was preincubated at 50°C. for 30 min and the reaction initiated by addition of 1-propanol([final]=600 mM). At least five samples over a period of 1 to 6 minuteswere taken, limiting overall substrate conversions to <5%. The sampleswere analyzed by GC, using a HP-1 column (length 30 m, i.d. 0.32 mm,film 0.25 mm) connected to a FID. Hydrogen was used as the carrier gas.For 4, the GC program ran isothermal at 110° C. for 10 min. Theretention time for substrate and propyl-ester was 5.4 min and 7.4 minrespectively. For 5, the GC program ran isothermal at 170° C. for 30min. The retention time for substrate and propyl-ester was 10.4 min and12.8 min respectively.

1. A circularly permuted protein comprising: lipase B from CandidaAntarctica (CALB); a linker sequence linking a native amino-terminal endof CALB and a native carboxy-terminal end of CALB; and a newamino-terminal end and a new carboxy-terminal end, wherein the newamino-terminal and carboxy-terminal ends of the circularly permutedprotein are different from the native amino-terminal andcarboxy-terminal ends of a corresponding native CALB protein, whereinthe new amino-terminal end is located at an amino acid residuecorresponding to an amino acid residue of the CALB protein SEQ ID No: 2selected from: 144, 148, 150, 268, 277, 278, 283, 284, 289, and 294,wherein the circularly permuted protein comprises at least oneimprovement over the corresponding native CALB protein, wherein theimprovement is selected from: increased activity, increasedaccessibility to the active site, increased flexibility of the activesite, increased the enantioselectivity, and broader or changed substratespecificity.
 2. The circularly permuted protein of claim 1, wherein thecircularly permuted protein has an active site and wherein, when thecircularly permuted protein is in a folded confirmation, the new aminoterminal and carboxy terminal ends are located near the active site ofthe protein.
 3. The circularly permuted protein of claim 1, wherein thecircularly permuted protein has increased accessibility to the activesite over that of the native protein.
 4. The circularly permuted proteinof claim 3, wherein the increased active site accessibility allows thecircularly permuted protein to couple to at least one substrate that thecorresponding native protein is substantially unable to couple.
 5. Thecircularly permuted protein of claim 3, wherein the increased activesite accessibility broadens or changes the substrate specificity of thecircularly permuted protein over that of the corresponding nativeprotein.
 6. The circularly permuted protein of claim 1, wherein thecircularly permuted protein has increased flexibility to the active siteas compared to that of the corresponding native protein.
 7. Thecircularly permuted protein of claim 1, wherein the circularly permutedprotein has substantially similar or increased enantioselectivity overthat of the corresponding native protein.
 8. The circularly permutedprotein of claim 1, wherein the circularly permuted protein hasincreased activity over that of the corresponding native protein.
 9. Thecircularly permuted protein of claim 1, wherein the protein isimmobilized to a surface.
 10. The circularly permuted protein of claim9, wherein the protein is immobilized to a matrix material.
 11. Thecircularly permuted protein of claim 9, wherein the immobilizationincreases the stability of the circularly permuted protein.
 12. Acircularly permuted protein comprising: lipase B from Candida Antarctica(CALB) having an original amino acid sequence comprising the amino acidsequence of SEQ ID No: 2; a linker sequence linking an originalamino-terminal end of CALB and an original caboxy-terminal end of CALB;and a new amino-terminal end and a new carboxy-terminal end, wherein thenew amino-terminal and carboxy-terminal ends of the circularly permutedprotein of the α/β-hydrolase fold family are different from the originalamino-terminal and carboxy-terminal ends of a corresponding native CALBprotein, wherein the new amino-terminal end is located in a region ofthe corresponding native CALB protein selected from: α7, α9, α16 andα17; wherein the circularly permuted protein comprises at least oneimprovement over the corresponding native CALB protein, wherein theimprovement is selected from: increased activity, increasedaccessibility to the active site, increased flexibility of the activesite, increased the enantioselectivity, and broader or changed substratespecificity.
 13. The circularly permuted protein of claim 12, whereinthe circularly permuted protein has an active site and wherein, when thecircularly permuted protein is in a folded confirmation, the new aminoterminal and carboxy terminal ends are located near the active site. 14.The circularly permuted protein of claim 12, wherein the circularlypermuted protein has a cap domain corresponding to a cap domain of thenative CALB protein and wherein, when the circularly permuted protein isin a folded confirmation, the new amino terminal and carboxy terminalends are located in the cap domain.
 15. The circularly permuted proteinof claim 12, wherein the protein has increased accessibility to theactive site over that of the native protein.
 16. The circularly permutedprotein of claim 15, wherein the increased active site accessibilityallows the circularly permuted protein to couple to at least onesubstrate that the corresponding native protein is substantially unableto couple.
 17. The circularly permuted protein of claim 15, wherein theincreased active site accessibility allows the circularly permutedprotein to couple esters and amides.
 18. The circularly permuted proteinof claim 17, wherein the esters are selected from: esters of primaryalcohols, esters of secondary alcohols, and esters of tertiary alcohols.19. The circularly permuted protein of claim 12, wherein the protein hasincreased flexibility to the active site as compared to that of thecorresponding native protein.
 20. The circularly permuted protein ofclaim 12, wherein the protein has substantially similar or increasedenantioselectivity over that of the corresponding native protein. 21.The circularly permuted protein of claim 12, wherein the protein hasincreased activity over that of the corresponding native protein. 22.The circularly permuted protein of claim 12, wherein the protein isimmobilized to a surface.
 23. The circularly permuted protein of claim12, wherein the new amino-terminal end is located at a residuecorresponding to an amino acid residue of SEQ ID No: 2 selected from:144, 148, 150, 193, 268, 277, 278, 283, 284, 289, and
 294. 24. Thecircularly permuted protein of claim 12, wherein the new amino-terminalend is located at a residue corresponding to residue 283 of SEQ ID No:2.
 25. The circularly permuted protein of claim 12, wherein thecircularly permuted protein is cp283 having SEQ ID No: 14, wherein cp283comprises an external loop region comprising the original amino-terminaland carboxy-terminal ends of the corresponding native protein and thelinker sequence, and wherein the secondary mutation comprises a deletionof one or more amino acids in the external loop region.
 26. Thecircularly permuted protein of claim 25, wherein the deletion of one ormore amino acids in the external loop region results in a secondcircularly permuted protein, and wherein the second circularly permutedprotein has substantially similar or increased activity over cp283 andhas increased stability over cp283.
 27. A circularly permuted proteincomprising: lipase B from Candida Antarctica (CALB), wherein an originalamino-terminal end and an original carboxy-terminal end of CALB havebeen linked together; a new amino-terminal end and a newcarboxy-terminal end, wherein the new amino-terminal andcarboxy-terminal ends of the circularly permuted protein are differentfrom the original amino-terminal and carboxy-terminal ends of acorresponding native CALB protein, wherein the new amino-terminal end islocated at an amino acid residue corresponding to an amino acid residueof SEQ ID No: 2 selected from: 144, 148, 150, 268, 277, 278, 283, 284,289, and 294, wherein the circularly permuted protein comprises at leastone improvement over the corresponding native CALB protein, wherein theimprovement is selected from: increased activity, increasedaccessibility to the active site, increased flexibility of the activesite, increased the enantioselectivity, and broader or changed substratespecificity.
 28. The circularly permuted protein of claim 1, wherein thecircularly permuted protein is cp283 comprising SEQ ID No:
 14. 29. Thecircularly permuted protein of claim 1, wherein the new amino-terminalend is located at a residue corresponding to residue 283 of SEQ ID NO:2.
 30. The circularly permuted protein of claim 29, wherein thecircularly permuted protein is cp283 having SEQ ID No: 14, wherein cp283comprises an external loop region comprising the original amino-terminaland carboxy-terminal ends of the corresponding native protein and thelinker sequence, and wherein the circularly permuted protein furthercomprises a secondary mutation comprising a deletion of one or moreamino acids in the external loop region.
 31. The circularly permutedprotein of claim 30, wherein the deletion of one or more amino acids inthe external loop region results in a second circularly permutedprotein, and wherein the second circularly permuted protein hassubstantially similar or increased activity over cp283 and has increasedstability over cp283.